Proteases comprising one or more combinable mutations

ABSTRACT

The present invention provides engineered protease variants. In particular, the protease variants comprise combinable mutations at selected surface positions that affect the charge and/or hydrophobicity of the enzyme to enhance at least one desired property of the resulting variant enzyme in a chosen application. Compositions comprising the protease variants, and methods for using the same are also provided.

The present application is a divisional application of U.S. patentapplication Ser. No. 13/128,854, filed Aug. 1, 2011, now U.S. Pat. No.8,753,861 which is a U.S. National Phase Application of InternationalApplication No. PCT/US20091063837, filed Nov. 10, 2009, which claimspriority to US Provisional Patent Application Ser. No. 61/113,545, filedon Nov. 11, 2008 and US Provisional Patent Application Ser. No.61/218,802, filed on Jun. 19, 2009, which are herein incorporated byreference in their entireties.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R.§1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “NB31065-US-PCD-SEQLIST.txt”created on Apr. 30, 2014, which is 19,284 bytes in size.

FIELD OF THE INVENTION

The present invention provides engineered protease variants. Inparticular, the protease variants comprise combinable mutations atselected surface positions that affect the charge and/or hydrophobicityof the enzyme to enhance at least one desired property of the resultingvariant enzyme in a chosen application. Compositions comprising theprotease variants, and methods for using the same are also provided.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolases comprising adiverse class of enzymes having a wide range of specificities andbiological functions. Much research has been conducted on thesubtilisins, due largely to their usefulness in cleaning and feedapplications. Additional work has been focused on the adverseenvironmental conditions (e.g., exposure to oxidative agents, chelatingagents, extremes of temperature and/or pH) that can diminish thefunctionality of these enzymes in various applications. Nonetheless,there remains a need in the art for enzyme systems that are able toresist these adverse conditions and retain or have improved activityover those currently known in the art.

SUMMARY OF THE INVENTION

The present invention provides engineered protease variants. Inparticular, the protease variants comprise combinable mutations atselected surface positions that affect the charge and/or hydrophobicityof the enzyme to enhance at least one desired property of the resultingvariant enzyme in a chosen application. Compositions comprising theprotease variants, and methods for using the same are also provided.

In one embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin that hasproteolytic activity and comprises a substitution two or more positionsselected from positions 24, 45, 101, 109, 118, 213 and 217, wherein thepositions are numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin that hasproteolytic activity and comprises a substitution two or more positionsselected from positions 24, 45, 101, 109, 118, 213 and 217, wherein thepositions are numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1, and thathas a relative protein expression level performance index (TCA PI)and/or a stain removal activity performance index (BMI PI) that isgreater or equal to 0.5.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of Bacillus subtilisin GG36 that hasproteolytic activity and comprises two or more substitutions at two ormore positions selected from S24Q, S24E, S24L, S24R, R45Q, R45E, R45L,S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L,G118R, T213Q, T213L, T213R, T213E, L217Q, and L217E, wherein thepositions are numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of Bacillus subtilisin GG36 that hasproteolytic activity and comprises two or more substitutions at two ormore positions selected from: S24Q, S24E, S24L, S24R, R45Q, R45E, R45L,S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L,G118R, T213Q, T213L, T213R, T213E, L217Q, and L217E, wherein thepositions are numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1, and thathas a relative protein expression level performance index (TCA PI)and/or a stain removal activity performance index (BMI PI) that isgreater or equal to 0.5.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of Bacillus subtilisin FNA that hasproteolytic activity and comprises two or more substitutions at two ormore positions selected from S24Q, S24E, S24L, S24R, A45Q, A45E, A45L,A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L, N109R, K213Q,K213E, K213L, K213R, L217Q, and L217E, wherein the positions arenumbered by correspondence with the amino acid sequence of B.amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of Bacillus subtilisin FNA that hasproteolytic activity and comprises two or more substitutions at two ormore positions selected from S24Q, S24E, S24L, S24R, A45Q, A45E, A45L,A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L, N109R, K213Q,K213E, K213L, K213R, L217Q, and L217E, wherein the positions arenumbered by correspondence with the amino acid sequence of B.amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1, and that hasa relative protein expression level performance index (TCA PI) and/or astain removal activity performance index (BMI PI) that is greater orequal to 0.5.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin GG36 that hasproteolytic activity and comprises a combination of substitutionsselected from S24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q,S101Q-G118Q-T213Q, G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q,S24E-R45E-S101Q-G118Q-T213Q, S24E-R45E-S101E-G118Q-T213Q,S24E-R45E-S101E-Q109E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118E-T213Q,S24E-R45E-S101E-Q109E-G118E-T213E, S24L-R45Q-S101Q-G118Q-T213Q,S24L-R45L-S101Q-G118Q-T213Q, S24L-R45L-S101L-G118Q-T213Q,S24L-R45L-S101L-Q109L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118L-T213Q,S24L-R45L-S101L-Q109L-G118L-T213L, S24R-R45Q-S101Q-G118Q-T213Q,S24R-S101Q-G118Q-T213Q, S24R-S101R-G118Q-T213Q,S24R-S101R-Q109R-G118Q-T213Q, S24R-S101R-Q109R-G118R-T213Q,S24R-S101R-Q109R-G118R-T213R, S24E-S101R-Q109R-G118R-T213R,S24E-R45E-S101R-Q109R-G118R-T213R, S24E-R45E-S101E-Q109R-G118R-T213R,S24E-R45E-S101E-Q109E-G118R-T213R, S24E-R45E-S101E-Q109E-G118E-T213R,S24E-R45E-S101E-Q109E-G118E-T213E, S24Q-R45Q-S101Q-G118Q-T213Q-L217Q,and S24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions arenumbered by correspondence with the amino acid sequence of B.amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin GG36 that hasproteolytic activity and comprises the substitution T213Q, wherein saidposition is numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin FNA that hasproteolytic activity and a combination of substitutions selected fromS24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-N118Q-K213Q,S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q,S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q,S24E-A45E-S101E-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109E-N118Q-K213Q,S24E-A45E-S101E-N109E-N118E-K213Q, S24E-A45E-S101E-N109E-N118E-K213E,S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q,S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q,S24L-A45L-S101L-N109L-N118L-K213Q, S24L-A45L-S101L-N109L-N118L-K213L,S24R-A45Q-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q,S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K213Q,S24R-A45R-S101R-N109R-N118R-K213Q, S24R-A45R-S101R-N109R-N118R-K213R,S24E-A45R-S101R-N109R-N118R-K213R, S24E-A45E-S101R-N109R-N118R-K213R,S24E-A45E-S101E-N109R-N118R-K213R, S24E-A45E-S101E-N109E-N118R-K213R,S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E,S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, andS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positionscorrespond to the positions of BPN′ subtilisin of SEQ ID NO:1.

In another embodiment, the protease variant is the mature form of anisolated subtilisin variant of a Bacillus subtilisin FNA that hasproteolytic activity and comprises the substitution K213Q, wherein saidposition is numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1.

In another embodiment, the invention provides an isolated nucleic acidthat encodes any one of the protease variants described above.

In another embodiment, the invention provides an expression vector thatcomprises an isolated nucleic acid that encodes any one of the proteasevariants described above.

In another embodiment, the invention provides a host cell that comprisesan expression vector, which in turn comprises an isolated nucleic acidthat encodes any one of the protease variants described above.

In another embodiment, the invention provides a cleaning compositionthat comprises at least one protease variant that is the mature form ofan isolated subtilisin variant of a Bacillus subtilisin that hasproteolytic activity and comprises a substitution two or more positionsselected from positions 24, 45, 101, 109, 118, 213 and 217, wherein thepositions are numbered by correspondence with the amino acid sequence ofB. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1. In someembodiments, the cleaning composition is a detergent. In someembodiments, the detergent is a dish detergent. In other embodiments,the detergent is a laundry detergent e.g. heavy duty liquid or drylaundry detergent. In alternative embodiments, the cleaning compositionfurther comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin that has proteolytic activity andcomprises a substitution two or more positions selected from positions24, 45, 101, 109, 118, 213 and 217, wherein the positions are numberedby correspondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that has a relativeprotein expression level performance index (TCA PI) and/or a stainremoval activity performance index (BMI PI) that is greater or equal to0.5. In some embodiments, the cleaning composition is a detergent. Insome embodiments, the detergent is a dish detergent. In otherembodiments, the detergent is a laundry detergent e.g. heavy duty liquidor dry laundry detergent. In alternative embodiments, the cleaningcomposition further comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin GG36 that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L,S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L,T213R, T213E, L217Q, and L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin GG36 that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom: S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L,S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L,T213R, T213E, L217Q, and L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that has a relativeprotein expression level performance index (TCA PI) and/or a stainremoval activity performance index (BMI PI) that is greater or equal to0.5. In some embodiments, the cleaning composition is a detergent. Insome embodiments, the detergent is a dish detergent. In otherembodiments, the detergent is a laundry detergent e.g. heavy duty liquidor dry laundry detergent. In alternative embodiments, the cleaningcomposition further comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin FNA that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom S24Q, S24E, S24L, S24R, A45Q, A45E, A45L, A45R, S101Q, S101E,S101L, S101R, N109Q, N109E, N109L, N109R, K213Q, K213E, K213L, K213R,L217Q, and L217E, wherein the positions are numbered by correspondencewith the amino acid sequence of B. amyloliquefaciens subtilisin BPN′ setforth as SEQ ID NO:1, and that has a relative protein expression levelperformance index (TCA PI) and/or a stain removal activity performanceindex (BMI PI) that is greater or equal to 0.5. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin GG36 that has proteolytic activity andcomprises a combination of substitutions selected fromS24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q,G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-G118Q-T213Q,S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118Q-T213Q,S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E,S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q,S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q,S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L,S24R-R45Q-S101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q,S24R-S101R-G118Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q,S24R-S101R-Q109R-G118R-T213Q, S24R-S101R-Q109R-G118R-T213R,S24E-S101R-Q109R-G118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R,S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R,S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E,S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, andS24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin GG36 that has proteolytic activity andcomprises the substitution T213Q, wherein said position is numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the invention provides a cleaning compositionthat comprises at least one protease variant that is the mature form ofan isolated subtilisin variant of a Bacillus subtilisin FNA that hasproteolytic activity and comprises a combination of substitutionsselected from S24Q-A45Q-S101Q-N109Q-N118Q-K213Q,A45Q-S101Q-N109Q-N118Q-K213Q, S101Q-N109Q-N118Q-K213Q,N109Q-N118Q-K213Q, N118Q-K213Q, S24E-A45Q-S101Q-N109Q-N118Q-K213Q,S24E-A45E-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109Q-N118Q-K213Q,S24E-A45E-S101E-N109E-N118Q-K213Q, S24E-A45E-S101E-N109E-N118E-K213Q,S24E-A45E-S101E-N109E-N118E-K213E, S24L-A45Q-S101Q-N109Q-N118Q-K213Q,S24L-A45L-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109Q-N118Q-K213Q,S24L-A45L-S101L-N109L-N118Q-K213Q, S24L-A45L-S101L-N109L-N118L-K213Q,S24L-A45L-S101L-N109L-N118L-K213L, S24R-A45Q-S101Q-N109Q-N118Q-K213Q,S24R-A45R-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109Q-N118Q-K213Q,S24R-A45R-S101R-N109R-N118Q-K213Q, S24R-A45R-S101R-N109R-N118R-K213Q,S24R-A45R-S101R-N109R-N118R-K213R, S24E-A45R-S101R-N109R-N118R-K213R,S24E-A45E-S101R-N109R-N118R-K213R, S24E-A45E-S101E-N109R-N118R-K213R,S24E-A45E-S101E-N109E-N118R-K213R, S24E-A45E-S101E-N109E-N118E-K213R,S24E-A45E-S101E-N109E-N118E-K213E,S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, andS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positionscorrespond to the positions of BPN′ subtilisin of SEQ ID NO:1. In someembodiments, the cleaning composition is a detergent. In otherembodiments, the detergent is a dish detergent. In yet otherembodiments, the detergent is a laundry detergent e.g. heavy duty liquidor dry laundry detergent. In alternative embodiments, the cleaningcomposition further comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin FNA that has proteolytic activity andcomprises the substitution K213Q, wherein said position is numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin that has proteolytic activity andcomprises a substitution two or more positions selected from positions24, 45, 101, 109, 118, 213 and 217, wherein the positions are numberedby correspondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that further comprises oneor more additional enzymes or enzyme derivatives. The additional enzymesor enzyme derivatives are selected from hemicellulases, cellulases,peroxidases, proteases, metalloproteases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases, pectatelyases, mannanases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin that has proteolytic activity andcomprises a substitution two or more positions selected from positions24, 45, 101, 109, 118, 213 and 217, wherein the positions are numberedby correspondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that has a relativeprotein expression level performance index (TCA PI) and/or a stainremoval activity performance index (BMI PI) that is greater or equal to0.5, and that further comprises one or more additional enzymes or enzymederivatives. The additional enzymes or enzyme derivatives are selectedfrom hemicellulases, peroxidases, proteases, metalloproteases,cellulases, xylanases, lipases, phospholipases, esterases,perhydrolases, cutinases, pectinases, keratinases, reductases, oxidases,phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases,pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase,chondroitinase, laccase, and amylases, or mixtures thereof. In someembodiments, the cleaning composition is a detergent. In someembodiments, the detergent is a dish detergent. In other embodiments,the detergent is a laundry detergent e.g. heavy duty liquid or drylaundry detergent. In alternative embodiments, the cleaning compositionfurther comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin GG36 that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L,S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L,T213R, T213E, L217Q, and L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that further comprises oneor more additional enzymes or enzyme derivatives. The additional enzymesor enzyme derivatives are selected from hemicellulases, peroxidases,proteases, metalloproteases, cellulases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, the cleaningcomposition is a detergent. In some embodiments, the detergent is a dishdetergent. In other embodiments, the detergent is a laundry detergente.g. heavy duty liquid or dry laundry detergent. In alternativeembodiments, the cleaning composition further comprises at least onestabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin GG36 that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom: S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L,S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L,T213R, T213E, L217Q, and L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that has a relativeprotein expression level performance index (TCA PI) and/or a stainremoval activity performance index (BMI PI) that is greater or equal to0.5, and that further comprises one or more additional enzymes or enzymederivatives. The additional enzymes or enzyme derivatives are selectedfrom hemicellulases, peroxidases, proteases, metalloproteases,cellulases, xylanases, lipases, phospholipases, esterases,perhydrolases, cutinases, pectinases, keratinases, reductases, oxidases,phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases,pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase,chondroitinase, laccase, and amylases, or mixtures thereof. In someembodiments, the cleaning composition is a detergent. In someembodiments, the detergent is a dish detergent. In other embodiments,the detergent is a laundry detergent e.g. heavy duty liquid or drylaundry detergent. In alternative embodiments, the cleaning compositionfurther comprises at least one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of Bacillus subtilisin FNA that has proteolytic activity andcomprises two or more substitutions at two or more positions selectedfrom S24Q, S24E, S24L, S24R, A45Q, A45E, A45L, A45R, S101Q, S101E,S101L, S101R, N109Q, N109E, N109L, N109R, K213Q, K213E, K213L, K213R,L217Q, and L217E, wherein the positions are numbered by correspondencewith the amino acid sequence of B. amyloliquefaciens subtilisin BPN′ setforth as SEQ ID NO:1, and that has a relative protein expression levelperformance index (TCA PI) and/or a stain removal activity performanceindex (BMI PI) that is greater or equal to 0.5, and that furthercomprises one or more additional enzymes or enzyme derivatives. Theadditional enzymes or enzyme derivatives are selected fromhemicellulases, peroxidases, proteases, metalloproteases, cellulases,xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases,pectinases, keratinases, reductases, oxidases, phenol oxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, thecleaning composition is a detergent. In some embodiments, the detergentis a dish detergent. In other embodiments, the detergent is a laundrydetergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin GG36 that has proteolytic activity andcomprises a combination of substitutions selected fromS24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q,G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-G118Q-T213Q,S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118Q-T213Q,S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E,S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q,S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q,S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L,S24R-R45Q-S101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q,S24R-S101R-G118Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q,S24R-S101R-Q109R-G118R-T213Q, S24R-S101R-Q109R-G118R-T213R,S24E-S101R-Q109R-G118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R,S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R,S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E,S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, andS24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that further comprises oneor more additional enzymes or enzyme derivatives. The additional enzymesor enzyme derivatives are selected from hemicellulases, peroxidases,proteases, metalloproteases, cellulases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, the cleaningcomposition is a detergent. In some embodiments, the detergent is a dishdetergent. In other embodiments, the detergent is a laundry detergente.g. heavy duty liquid or dry laundry detergent. In alternativeembodiments, the cleaning composition further comprises at least onestabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin GG36 that has proteolytic activity andcomprises the substitution T213Q, wherein said position is numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that further comprises oneor more additional enzymes or enzyme derivatives. The additional enzymesor enzyme derivatives are selected from hemicellulases, peroxidases,proteases, metalloproteases, cellulases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, the cleaningcomposition is a detergent. In some embodiments, the detergent is a dishdetergent. In other embodiments, the detergent is a laundry detergente.g. heavy duty liquid or dry laundry detergent. In alternativeembodiments, the cleaning composition further comprises at least onestabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin FNA that has proteolytic activity andcomprises a combination of substitutions selected fromS24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-N118Q-K213Q,S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q,S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q,S24E-A45E-S101E-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109E-N118Q-K213Q,S24E-A45E-S101E-N109E-N118E-K213Q, S24E-A45E-S101E-N109E-N118E-K213E,S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q,S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q,S24L-A45L-S101L-N109L-N118L-K213Q, S24L-A45L-S101L-N109L-N118L-K213L,S24R-A45Q-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q,S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K213Q,S24R-A45R-S101R-N109R-N118R-K213Q, S24R-A45R-S101R-N109R-N118R-K213R,S24E-A45R-S101R-N109R-N118R-K213R, S24E-A45E-S101R-N109R-N118R-K213R,S24E-A45E-S101E-N109R-N118R-K213R, S24E-A45E-S101E-N109E-N118R-K213R,S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E,S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, andS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positionscorrespond to the positions of BPN′ subtilisin of SEQ ID NO:1, and thatfurther comprises one or more additional enzymes or enzyme derivatives.The additional enzymes or enzyme derivatives are selected fromhemicellulases, peroxidases, proteases, metalloproteases, cellulases,xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases,pectinases, keratinases, reductases, oxidases, phenol oxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, thecleaning composition is a detergent. In other embodiments, the detergentis a dish detergent. In yet other embodiments, the detergent is alaundry detergent e.g. heavy duty liquid or dry laundry detergent. Inalternative embodiments, the cleaning composition further comprises atleast one stabilizing agent.

In another embodiment, the cleaning composition comprises at least oneprotease variant that is the mature form of an isolated subtilisinvariant of a Bacillus subtilisin FNA that has proteolytic activity andcomprises the substitution K213Q, wherein said position is numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1, and that further comprises oneor more additional enzymes or enzyme derivatives. The additional enzymesor enzyme derivatives are selected from hemicellulases, peroxidases,proteases, metalloproteases, cellulases, xylanases, lipases,phospholipases, esterases, perhydrolases, cutinases, pectinases,keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, the cleaningcomposition is a detergent. In some embodiments, the detergent is a dishdetergent. In other embodiments, the detergent is a laundry detergente.g. heavy duty liquid or dry laundry detergent. In alternativeembodiments, the cleaning composition further comprises at least onestabilizing agent.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinthat has proteolytic activity and comprises a substitution two or morepositions selected from positions 24, 45, 101, 109, 118, 213 and 217,wherein the positions are numbered by correspondence with the amino acidsequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ IDNO:1.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinthat has proteolytic activity and comprises a substitution two or morepositions selected from positions 24, 45, 101, 109, 118, 213 and 217,wherein the positions are numbered by correspondence with the amino acidsequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ IDNO:1, and that has a relative protein expression level performance index(TCA PI) and/or a stain removal activity performance index (BMI PI) thatis greater or equal to 0.5.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of Bacillus subtilisinGG36 that has proteolytic activity and comprises two or moresubstitutions at two or more positions selected from S24Q, S24E, S24L,S24R, R45Q, R45E, R45L, S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109R, G118Q, G118E, G118L, G118R, T213Q, T213L, T213R, T213E, L217Q, andL217E, wherein the positions are numbered by correspondence with theamino acid sequence of B. amyloliquefaciens subtilisin BPN′ set forth asSEQ ID NO:1.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of Bacillus subtilisinGG36 that has proteolytic activity and comprises two or moresubstitutions at two or more positions selected from: S24Q, S24E, S24L,S24R, R45Q, R45E, R45L, S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109R, G118Q, G118E, G118L, G118R, T213Q, T213L, T213R, T213E, L217Q, andL217E, wherein the positions are numbered by correspondence with theamino acid sequence of B. amyloliquefaciens subtilisin BPN′ set forth asSEQ ID NO:1, and that has a relative protein expression levelperformance index (TCA PI) and/or a stain removal activity performanceindex (BMI PI) that is greater or equal to 0.5.

In another embodiment, the invention provides a cleaning compositioncomprising at least 0.0001 weight percent of at least one subtilisinvariant that is the mature form of an isolated subtilisin variant ofBacillus subtilisin FNA that has proteolytic activity and comprises twoor more substitutions at two or more positions selected from S24Q, S24E,S24L, S24R, A45Q, A45E, A45L, A45R, S101Q, S101E, S101L, S101R, N109Q,N109E, N109L, N109R, K213Q, K213E, K213L, K213R, L217Q, and L217E,wherein the positions are numbered by correspondence with the amino acidsequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ IDNO:1.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of Bacillus subtilisin FNAthat has proteolytic activity and comprises two or more substitutions attwo or more positions selected from S24Q, S24E, S24L, S24R, A45Q, A45E,A45L, A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L, N109R,K213Q, K213E, K213L, K213R, L217Q, and L217E, wherein the positions arenumbered by correspondence with the amino acid sequence of B.amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:1, and that hasa relative protein expression level performance index (TCA PI) and/or astain removal activity performance index (BMI PI) that is greater orequal to 0.5.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinGG36 that has proteolytic activity and comprises a combination ofsubstitutions selected from S24Q-R45Q-S101Q-G118Q-T213Q,R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q, G118Q-T213Q,S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-G118Q-T213Q,S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118Q-T213Q,S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E,S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q,S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q,S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L,S24R-R45Q-S101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q,S24R-S101R-G118Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q,S24R-S101R-Q109R-G118R-T213Q, S24R-S101R-Q109R-G118R-T213R,S24E-S101R-Q109R-G118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R,S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R,S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E,S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, andS24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions are numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN′ set forth as SEQ ID NO:1

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinGG36 that has proteolytic activity and comprises the substitution T213Q,wherein said position is numbered by correspondence with the amino acidsequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ IDNO:1.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinFNA that has proteolytic activity and comprises a combination ofsubstitutions selected from S24Q-A45Q-S101Q-N109Q-N118Q-K213Q,A45Q-S101Q-N109Q-N118Q-K213Q, S101Q-N109Q-N118Q-K213Q,N109Q-N118Q-K213Q, N118Q-K213Q, S24E-A45Q-S101Q-N109Q-N118Q-K213Q,S24E-A45E-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109Q-N118Q-K213Q,S24E-A45E-S101E-N109E-N118Q-K213Q, S24E-A45E-S101E-N109E-N118E-K213Q,S24E-A45E-S101E-N109E-N118E-K213E, S24L-A45Q-S101Q-N109Q-N118Q-K213Q,S24L-A45L-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109Q-N118Q-K213Q,S24L-A45L-S101L-N109L-N118Q-K213Q, S24L-A45L-S101L-N109L-N118L-K213Q,S24L-A45L-S101L-N109L-N118L-K213L, S24R-A45Q-S101Q-N109Q-N118Q-K213Q,S24R-A45R-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109Q-N118Q-K213Q,S24R-A45R-S101R-N109R-N118Q-K213Q, S24R-A45R-S101R-N109R-N118R-K213Q,S24R-A45R-S101R-N109R-N118R-K213R, S24E-A45R-S101R-N109R-N118R-K213R,S24E-A45E-S101R-N109R-N118R-K213R, S24E-A45E-S101E-N109R-N118R-K213R,S24E-A45E-S101E-N109E-N118R-K213R, S24E-A45E-S101E-N109E-N118E-K213R,S24E-A45E-S101E-N109E-N118E-K213E,S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, andS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positionscorrespond to the positions of BPN′ subtilisin of SEQ ID NO:1.

In another embodiment, the cleaning composition comprises at least0.0001 weight percent of at least one subtilisin variant that is themature form of an isolated subtilisin variant of a Bacillus subtilisinFNA that has proteolytic activity and comprises the substitution K213Q,wherein said position is numbered by correspondence with the amino acidsequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ IDNO:1.

In another embodiment, the invention provides a method of cleaning thatcomprises contacting a surface and/or an article comprising a fabricwith any one of the cleaning compositions described, and optionallywashing and/or rinsing said surface or article.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of the mature form of parent proteases GG36(SEQ ID:4) and FNA (SEQ ID NO:7) with BPN′ (SEQ ID NO:1). Unlessotherwise specified, substitution positions are given in relationship toBPN′

FIG. 2 provides a charge change matrix indicating the charge change foramino acid residue substitutions at pH 8.6. From this matrix the netcharge change of a variant enzyme as compared to a parent enzyme can beeasily determined.

FIG. 3 provides a Kyte-Doolittle hydropathicity change matrix indicatingthe hydropathicity change for amino acid residue substitutions. Fromthis matrix the net hydropathicity change of a variant enzyme ascompared to a parent enzyme can be easily determined.

FIG. 4 provides a map of pAC-GG36ci

FIG. 5 provides a map of pAC-FNAre.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered protease variants. Inparticular, the protease variants comprise combinable mutations atselected surface positions that affect the charge and/or hydrophobicityof the enzyme to enhance at least one desired property of the resultingvariant enzyme in a chosen application. Compositions comprising theprotease variants, and methods for using the same are also provided.

As indicated herein, introducing substitutions that affect the chargeand/or hydrophobicity of subtilisin results in an enzyme that comprisesat least one combinable mutation to provide a variant protease having aPerformance Index for at least one property of interest being aperformance index >0.5 when compared to that of the parent enzyme. Thecombinable mutations serve to enhance the performance index for at leastone desired enzyme property in a variety of applications. Properties ofinterest include but are not limited to charge, hydrophobicity,solubility, cleaning performance e.g. stain removal from fabric and/orhard surfaces, thermal stability, storage stability, detergentstability, substrate binding, enzyme inhibition, expression level,reaction rate, and substrate degradation. In some embodiments, theproperty of interest is one or more properties selected from charge,hydrophobicity, expression level (TCA PI) and cleaning performance e.g.stain removal (BMI PI). Although described herein in relationship toproteases and blood, milk and ink stains (BMI), it is contemplated thatthe protease variants of the present invention are optimized for anyenzyme-substrate interaction in any a variety of reaction media dictatedby the application e.g. cleaning applications.

Previously, efforts to develop superior proteins focused upon minimizingenzyme binding to surfaces. For example, some methods involved alteringthe subtilisin sequence to obtain variant enzymes with decreasedadsorption to insoluble substrates (See e.g., WO 95/07991). In anotherapproach, the pI of subtilisin was altered in order to obtain variantenzymes with a net charge of zero at a defined pH (See e.g., WO91/00345). However, as determined during development of the presentinvention, these approaches are not always successful. During thedevelopment of the present invention, it was determined that surfaceproperties of enzymes generally have optima that are determined as afunction of change in surface charge and/or hydrophobicity. Even forenzymes that are normally quite active, surface properties can cause theoverall reaction to be much slower under some conditions and with somesubstrates than under other conditions and/or with other substrates. Insome embodiments the present invention provides variant proteases thatcomprise modified surface properties obtained by changing the nature ofone or more amino acids on the enzyme surface. When these changes aremade at sites on the surface that do not interact with any other aminoacids and are not necessary for enzyme function, the properties of theprotein are predicted based on the properties of the amino acidssubstituted at those positions as described herein.

Sites are readily identified from structure data; alternatively,homologous sequence alignments, site evaluation library data and/or anycombination thereof find use. Amino acid scoring matrices (e.g. FIG. 1)and/or hydrophobicity scales (e.g. FIG. 2) find use in guiding aminoacid substitution(s) and to identify those physical properties of theprotein that correlate with the properties of the substituted aminoacids.

Definitions

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works well known to thoseskilled in the art. All patents, patent applications, articles andpublications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any methodsand materials similar or equivalent to those described herein find usein the practice of the present invention, some of the preferred methodsand materials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to theSpecification as a whole.

Also, as used herein, the singular “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Numericranges are inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention, which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

As used herein, there term “performance index (PI)” refers to the ratioof the performance of a variant enzyme relative to that of the parent orreference enzyme in a given assay. The PI for cleaning performance isprovided herein as the performance to remove blood, milk, and ink stains(BMI), and is provided as a BMI PI value. The PI for expression levelperformance is provided herein as the level of protein produced asmeasured by trichloroacetic (TCA) acid precipitation, and is provided asa TCA PI value.

As used herein, “combinable mutations” are those mutations for which thevariant comprising the mutations has Performance Index (PI) a value >0.5for at least one property. Combinable mutations are mutations that canbe combined to deliver proteins with appropriate Performance Indices forone or more desired properties, and have changes in charge and/orhydrophobicity. Positions at which mutations occur are classed asfollows: Non-restrictive positions have=20% neutral mutations for atleast one property; and Restrictive positions have <20% neutralmutations for activity and stability.

The term “isolated” or “purified” refers to a material that is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, the material is said to be “purified”when it is present in a particular composition in a higher or lowerconcentration than exists in a naturally occurring or wild type organismor in combination with components not normally present upon expressionfrom a naturally occurring or wild type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. In some embodiments, such polynucleotides are partof a vector, and/or such polynucleotides or polypeptides are part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. In preferred embodiments, a nucleicacid or protein is said to be purified, for example, if it gives rise toessentially one band in an electrophoretic gel or blot.

The term “isolated”, when used in reference to a DNA sequence, refers toa DNA sequence that has been removed from its natural genetic milieu andis thus free of other extraneous or unwanted coding sequences, and is ina form suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]).The term “an isolated DNA sequence” is alternatively referred to as “acloned DNA sequence”.

The term “isolated,” when used in reference to a protein, refers to aprotein that is found in a condition other than its native environment.In a preferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins. An isolated protein ismore than about 10% pure, preferably more than about 20% pure, and evenmore preferably more than about 30% pure, as determined by SDS-PAGE.Further aspects of the invention encompass the protein in a highlypurified form (i.e., more than about 40% pure, more than about 60% pure,more than about 70% pure, more than about 80% pure, more than about 90%pure, more than about 95% pure, more than about 97% pure, and even morethan about 99% pure), as determined by SDS-PAGE.

As used herein, a “parent” protein refers to the protein that ismodified e.g. by introducing one or more amino acid substitutions, togenerate one or more variant of the parent protein. Thus, the terms“protease variant” and “variant protease” are used in reference toparent proteases that are similar to variant protease, particularly intheir function, but have mutations in their amino acid sequence thatmake them different in sequence from the parent protease at from one to20 amino acid positions. The amino acid sequences of the mature regionof exemplary parent proteases are shown in the alignment of FIG. 1. FNA(SEQ ID NO:7) and GG36 (SEQ ID NO:4) are the mature parent proteaseswhich have been modified to contain one or more combinable substitutionsto generate the variant proteases of the invention. GG36 is a wild-typeBacillus lentus protease, and FNA is the Bacillus amyloliquefaciens BPN′protease containing the Y217L substitution.

As used herein, the terms “protease,” and “proteolytic activity” referto a protein or peptide exhibiting the ability to hydrolyze peptides orsubstrates having peptide linkages. Many well known procedures exist formeasuring proteolytic activity (Kalisz, “Microbial Proteinases,” In:Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology,[1988]). For example, proteolytic activity may be ascertained bycomparative assays which analyze the respective protease's ability tohydrolyze a commercial substrate. Exemplary substrates useful in theanalysis of protease or proteolytic activity, include, but are notlimited to di-methyl casein (Sigma C-9801), bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICNBiomedical 902111). Colorimetric assays utilizing these substrates arewell known in the art (See e.g., WO 99/34011; and U.S. Pat. No.6,376,450, both of which are incorporated herein by reference). The pNAassay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) alsofinds use in determining the active enzyme concentration for fractionscollected during gradient elution. This assay measures the rate at whichp-nitroaniline is released as the enzyme hydrolyzes the solublesynthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(suc-AAPF-pNA). The rate of production of yellow color from thehydrolysis reaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nm can be used to determine the total proteinconcentration. The active enzyme/total-protein ratio gives the enzymepurity.

As used herein, the term “subtilisin” refers any member of the S8 serineprotease family as described in MEROPS—The Peptidase Data base (Rawlingset al., MEROPS: the peptidase database, Nucleic Acids Res, 34 Databaseissue, D270-272, 2006, at the websitemerops.sanger.ac.uk/cgi-bin/merops.cgi?id=s08;action=.). The followinginformation was derived from MEROPS—The Peptidase Data base as of Nov.6, 2008 “Peptidase family S8 contains the serine endopeptidasesubtilisin and its homologues (Biochem J, 290:205-218, 1993). Family S8,also known as the subtilase family, is the second largest family ofserine peptidases, and can be divided into two subfamilies, withsubtilisin (S08.001) the type-example for subfamily S8A and kexin(S08.070) the type-example for subfamily S8B. Tripeptidyl-peptidase II(TPP-II; 508.090) was formerly considered to be the type-example of athird subfamily, but has since been determined to be misclassified.Members of family S8 have a catalytic triad in the order Asp, His andSer in the sequence, which is a different order to that of families S1,S9 and S10. In subfamily S8A, the active site residues frequently occursin the motifs Asp-Thr/Ser-Gly (which is similar to the sequence motif infamilies of aspartic endopeptidases in clan AA), His-Gly-Thr-His andGly-Thr-Ser-Met-Ala-Xaa-Pro. In subfamily S8B, the catalytic residuesfrequently occur in the motifs Asp-Asp-Gly, His-Gly-Thr-Arg andGly-Thr-Ser-Ala/Val-Ala/Ser-Pro. Most members of the S8 family areendopeptidases, and are active at neutral-mildly alkali pH. Manypeptidases in the family are thermostable. Casein is often used as aprotein substrate and a typical synthetic substrate is suc-AAPF. Mostmembers of the family are nonspecific peptidases with a preference tocleave after hydrophobic residues. However, members of subfamily S8B,such as kexin (S08.070) and furin (S08.071), cleave after dibasic aminoacids. Most members of the S8 family are inhibited by general serinepeptidase inhibitors such as DFP and PMSF. Because many members of thefamily bind calcium for stability, inhibition can be seen with EDTA andEGTA, which are often thought to be specific inhibitors ofmetallopeptidases. Protein inhibitors include turkey ovomucoid thirddomain (I01.003), Streptomyces subtilisin inhibitor (I16.003), andmembers of family I13 such as eglin C (I13.001) and barley inhibitorCI-1A (I13.005), many of which also inhibit chymotrypsin (S01.001). Thesubtilisin propeptide is itself inhibitory, and the homologousproteinase B inhibitor from Saccharomyces inhibits cerevisin (S08.052).The tertiary structures for several members of family S8 have now beendetermined. A typical S8 protein structure consists of three layers witha seven-stranded β sheet sandwiched between two layers of helices.Subtilisin (S08.001) is the type structure for clan SB (SB). Despite thedifferent structure, the active sites of subtilisin and chymotrypsin(S01.001) can be superimposed, which suggests the similarity is theresult of convergent rather than divergent evolution.

As used herein, the terms “Bacillus” and “genus Bacillus” include allspecies within the genus “Bacillus,” as known to those of skill in theart, including but not limited to B. subtilis, B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.coagulans, B. circulans, B. lautus, and B. thuringiensis. It isrecognized that the genus Bacillus continues to undergo taxonomicalreorganization. Thus, it is intended that the genus include species thathave been reclassified, including but not limited to such organisms asB. stearothermophilus, which is now named “GeoBacillusstearothermophilus.” The production of resistant endospores in thepresence of oxygen is considered the defining feature of the genusBacillus, although this characteristic also applies to the recentlynamed AlicycloBacillus, AmphiBacillus, AneuriniBacillus, AnoxyBacillus,BreviBacillus, FiloBacillus, GraciliBacillus, HaloBacillus,PaeniBacillus, SaliBacillus, ThermoBacillus, UreiBacillus, andVirgiBacillus.

The terms “protein” and “polypeptide” are used interchangeabilityherein. The 3-letter code for amino acids as defined in conformity withthe IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) isused through out this disclosure. It is also understood that apolypeptide may be coded for by more than one nucleotide sequence due tothe degeneracy of the genetic code.

A “prosequence” is an amino acid sequence between the signal peptide andthe mature region of a protease. The prosequence is cleaved during thematuration process that results in the production of the active matureform of the protease.

The term “signal sequence” or “signal peptide” refers to any sequence ofnucleotides and/or amino acids that participate in the secretion of themature or precursor forms of the protein. This definition of signalsequence is a functional one, meant to include all those amino acidsequences encoded by the N-terminal portion of the protein gene, whichparticipate in the effectuation of the secretion of protein. They areoften, but not universally, bound to the N-terminal portion of a proteinor to the N-terminal portion of a precursor protein. The signal sequencemay be endogenous or exogenous. The signal sequence may be that normallyassociated with the protein (e.g., protease), or may be from a geneencoding another secreted protein. One exemplary exogenous signalsequence comprises the first seven amino acid residues of the signalsequence from B. subtilis subtilisin fused to the remainder of thesignal sequence of the subtilisin from B. lentus (ATCC 21536).

The term “hybrid signal sequence” refers to signal sequences in whichpart of sequence is obtained from the expression host fused to thesignal sequence of the gene to be expressed. In some embodiments,synthetic sequences are utilized.

The term “mature” form of a protein or peptide refers to the finalfunctional form of the protein or peptide. To exemplify, the mature formof the FNA protease provided herein includes the amino acid sequence ofSEQ ID NO:7, while a mature form of the GG36 protease includes the aminoacid sequence of SEQ ID NO:4.

The term “precursor” herein refers to the form of a protein or peptidehaving a prosequence operably linked to the amino or carbonyl terminusof the mature protein. To exemplify, SEQ ID NOS:3 and 6 are sequences ofthe precursors of the mature GG36 (SEQ ID NO:4) and FNA (SEQ ID NO:7),respectively. The precursor may also have a “signal” sequence operablylinked, to the amino terminus of the prosequence. The precursor may alsohave additional polynucleotides that are involved in post-translationalactivity (e.g., polynucleotides cleaved therefrom to leave the matureform of a protein or peptide).

“Naturally occurring enzyme” and “naturally occurring protein” refer toan enzyme or protein having the unmodified amino acid sequence identicalto that found in nature. Naturally occurring enzymes include nativeenzymes, those enzymes naturally expressed or found in the particularmicroorganism.

The terms “derived from” and “obtained from” refer to not only an enzyme(e.g., protease) produced or producible by a strain of the organism inquestion, but also an enzyme encoded by a DNA sequence isolated fromsuch strain and produced in a host organism containing such DNAsequence. Additionally, the term refers to a enzyme that is encoded by aDNA sequence of synthetic and/or cDNA origin and which has theidentifying characteristics of the enzyme in question.

A “derivative” within the scope of this definition generally retains thecharacteristic proteolytic activity observed in the wild-type, native orparent form to the extent that the derivative is useful for similarpurposes as the wild-type, native or parent form. Functional enzymederivatives encompass naturally occurring, synthetically orrecombinantly produced peptides or peptide fragments having the generalcharacteristics of the parent enzyme.

As used herein, “by correspondence to” refers to a residue at theenumerated position in a protein or peptide.

As used herein, “substituted” and “substitutions” refer toreplacement(s) of one or more amino acid residues or nucleic acid basesin a parent sequence. In some embodiments, the substitution involves thereplacement of a naturally occurring residue or base. In someembodiments, two or more amino acids are substituted to generate avariant protease that comprises a combination of amino acidsubstitutions. In some embodiments, combinations of substitutions aredenoted by the amino acid position at which the substitution is made.For example, a combination denoted by E6A-E30G means that glutamic acid(E) at position 6 is substituted with Alanine (A) and the glutamic acid(E) at position 30 is substituted with a Glycine (G). Amino acidpositions are given by correspondence to the numbered position in themature region of the subtilisin BPN′ (SEQ ID NO:1).

The phrase “substitution at two or more positions” herein refers to acombination of two or more substitutions that are made in the sameprotein. Thus, “a substitution at two or more positions” refers to anyone of a combination of 2, 3, 4, 5, 6, and 7 amino acid substitutions.

As used herein, the terms “expression cassette” and “expression vector”refer to nucleic acid constructs generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a target cell. Therecombinant expression cassette can be incorporated into a plasmid,chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acidfragment. Typically, the recombinant expression cassette portion of anexpression vector includes, among other sequences, a nucleic acidsequence to be transcribed and a promoter. In preferred embodiments,expression vectors have the ability to incorporate and expressheterologous DNA fragments in a host cell. Many prokaryotic andeukaryotic expression vectors are commercially available. Selection ofappropriate expression vectors is within the knowledge of those of skillin the art. The term “expression cassette” is used interchangeablyherein with “DNA construct,” and their grammatical equivalents.Selection of appropriate expression vectors is within the knowledge ofthose of skill in the art.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,cassettes and the like. In some embodiments, the polynucleotideconstruct comprises a DNA sequence encoding the protease (e.g.,precursor or mature protease) that is operably linked to a suitableprosequence (e.g., secretory, etc.) capable of effecting the expressionof the DNA in a suitable host.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orprokaryotes, or integrates into the host chromosome.

As used herein, the terms “Host strain” or “host cell” refers to asuitable host for an expression vector comprising DNA according to thepresent invention.

As used herein, “cleaning compositions” and “cleaning formulations”refer to compositions that find use in the removal of undesiredcompounds from items to be cleaned, such as fabric, dishes, contactlenses, other solid substrates, hair (shampoos), skin (soaps andcreams), teeth (mouthwashes, toothpastes) etc. The term encompasses anymaterials/compounds selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, powder or spray composition), used in the composition. Thespecific selection of cleaning composition materials are readily made byconsidering the surface, item or fabric to be cleaned, and the desiredform of the composition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning,bleaching, disinfecting, and/or sterilizing any object and/or surface.It is intended that the terms include, but are not limited to detergentcompositions (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unlessotherwise indicated, dry e.g. granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid, gelor paste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, “non-fabric cleaning compositions” include non-textile(i.e., fabric) surface cleaning compositions, including but not limitedto dishwashing detergent compositions, oral cleaning compositions,denture cleaning compositions, and personal cleansing compositions.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In preferredembodiments, the term is used in reference to detergents used to cleandishes, cutlery, etc. (e.g., “dish detergents” or “dishwashingdetergents”). It is not intended that the present invention be limitedto any particular detergent formulation or composition. Indeed, it isintended that in addition to detergents that contain at least oneprotease of the present invention, the term encompasses detergents thatcontain surfactants, transferase(s), hydrolytic enzymes, oxidoreductases, builders, bleaching agents, bleach activators, bluing agentsand fluorescent dyes, caking inhibitors, masking agents, enzymeactivators, antioxidants, and solubilizers.

As used herein, “dishwashing composition” refers to all forms ofcompositions for cleaning dishware, including cutlery, including but notlimited to granular and liquid forms. It is not intended that thepresent invention be limited to any particular type or dishwarecomposition. Indeed, the present invention finds use in cleaningdishware (e.g., dishes, including, but not limited to plates, cups,glasses, bowls, etc.) and cutlery (e.g., utensils, including but notlimited to spoons, knives, forks, serving utensils, etc.) of anymaterial, including but not limited to ceramics, plastics, metals,china, glass, acrylics, etc. The term “dishware” is used herein inreference to both dishes and cutlery.

As used herein, “non-phosphate containing dishwashing detergents” aredetergents that contain no more than 0.5% phosphorus (i.e., phosphorusis a trace element).

As used herein, “wash performance” or “cleaning performance” of avariant protease refers to the contribution made by a variant proteaseto the cleaning ability of a cleaning composition when compared to thatobtained in the absence of the protease variant.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,sud concentration, type of detergent and water hardness, actually usedin households in a dish or laundry detergent market segment.

The term “improved wash performance” is used to indicate that a betterend result is obtained in stain removal under relevant washingconditions, or that less variant protease, on weight basis, is needed toobtain the same end result relative to the corresponding wild-type orstarting parent protease.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentinvention be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

As used herein, “effective amount of enzyme” refers to the quantity ofenzyme necessary to achieve the enzymatic activity required in thespecific application (e.g., personal care product, cleaning composition,etc.). Such effective amounts are readily ascertained by one of ordinaryskill in the art and are based on many factors, such as the particularenzyme variant used, the cleaning application, the specific compositionof the cleaning composition, and whether a liquid or dry (e.g.,granular, bar) composition is required, and the like.

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically about 17 to about 35% by weight of the total composition. Incontrast, in compact compositions, the filler salt is present in amountsnot exceeding about 15% of the total composition. In some embodiments,the filler salt is present in amounts that do not exceed about 10%, ormore preferably, about 5%, by weight of the composition. In someembodiments, the inorganic filler salts are selected from the alkali andalkaline-earth-metal salts of sulfates and chlorides. A preferred fillersalt is sodium sulfate.

As used herein, “adjunct ingredient” or “adjunct material” refers tocleaning materials that include, but are not limited to, surfactants,builders, bleaches, bleach activators, bleach catalysts, other enzymes,enzyme stabilizing systems, chelants, optical brighteners, soil releasepolymers, dye transfer agents, dispersants, suds suppressors, dyes,perfumes, colorants, filler salts, hydrotropes, photoactivators,fluorescers, fabric conditioners, hydrolyzable surfactants,preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkleagents, germicides, fungicides, color speckles, silvercare, anti-tarnishand/or anti-corrosion agents, alkalinity sources, solubilizing agents,carriers, processing aids, pigments, and pH control agents (See e.g.,U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504,5,695,679, 5,686,014 and 5,646,101, all of which are incorporated hereinby reference).

As used herein, “dishwashing composition” refers to all forms forcompositions for cleaning dishes, including but not limited to granularand liquid forms.

As used herein, “fabric cleaning composition” refers to all forms ofdetergent compositions for cleaning fabrics, including but not limitedto, granular, liquid and bar forms.

As used herein, “fabric” encompasses any textile material. Thus, it isintended that the term encompass garments, as well as fabrics, yarns,fibers, non-woven materials, natural materials, synthetic materials, andany other textile material.

As used herein, “textile” refers to woven fabrics, as well as staplefibers and filaments suitable for conversion to or use as yarns, woven,knit, and non-woven fabrics. The term encompasses yarns made fromnatural, as well as synthetic (e.g., manufactured) fibers.

As used herein, “textile materials” is a general term for fibers, yarnintermediates, yarn, fabrics, and products made from fabrics (e.g.,garments and other articles).

Most strategies currently utilized for improving protein performance inindustrial, consumer or pharmaceutical applications have focused onamino acid substitutions at or near an enzyme's active site, in order toincrease catalytic efficiency. However, during the development of thepresent invention, it was determined that mutations elsewhere on theenzyme surface dramatically increase enzyme performance beyond what ispossible through catalytic efficiency improvements. Basically, thereaction rate governing conversion of substrates to products mediated byenzymes is only partially controlled by the rate of the chemicalcatalytic conversion step alone. Enzymes and substrates interact ascolloids prior to their association as an enzyme-substrate ES complex,as well as during dissociation from the enzyme-product EP complex thatis formed after chemical conversion. Even if the reaction step proceedsat a fast rate, enzyme approach towards substrate can be extremely slow(e.g., diffusion-limited), as in the case of same-sign colloidsexperiencing electrostatic repulsive forces. Likewise, release of enzymefrom the enzyme-product EP complex can be extremely slow (e.g.,diffusion-limited), as in the case of colloids experiencing attractiveshort-range hydrophobic and dispersive forces. Both conditions increasethe enzyme transit time from substrate to product and become rate-steplimiting compared to chemical conversion. While it is possible toenvisage that oppositely-charged colloids would actually accelerate theformation of ES complexes (e.g., above the diffusion limit), subsequentdissociation of the EP complex would be painfully slow (assuming that nocharges are created nor lost) and the overall reaction rate woulddecrease. Therefore, the asymmetry of the pair-wise interactionpotential is exploited in order to ensure minimal transit times for boththe ES and EP complexes. This is particularly important in industrialbiotechnology, since it is desirable to convert all of the substrate toproduct in the shortest amount of time possible under oftenenzyme-limited conditions. Historically, protein engineers have focusedon specific enzyme-substrate interactions of the chemical conversionstep and have failed to recognize the contribution of both short- andlong-range non-specific interactions, arising from intermolecularcolloidal and surface forces, which govern the association anddissociation steps. An objective of the present invention is to provideprotease variants having altered surface properties obtained by changingthe nature of one or more amino acids on the enzyme surface thatoptimize intermolecular forces to the point where the chemicalconversion step becomes rate-limiting.

Modification of surface properties is achieved by introducing amino acidsubstitutions that alter the charge and/or the hydrophobicity of theenzyme using the methods described herein. Once the chemical conversionstep becomes rate limiting, further improvements in the performance ofthe variant enzyme can be achieved through changes in the enzyme activesite. This objective is applicable whether the substrate is a smallpeptide in solution or an insoluble macroscopic substrate. Nonetheless,knowledge of the mechanism(s) involved is not necessary in order to makeand use the present invention. Nor is it intended that the presentinvention be limited to any particular mechanism.

Briefly the methods used to generate the protease variants of thepresent invention involve (I) Assaying Probe Proteins Spanning aPhysical Property Range; (II) Determining Physical Property Optimum fora Given Favorable Outcome; and (III) Providing Variant Proteins HavingThe Physical Property Optimum.

I. Assay Probe Proteins

Assaying probe proteins involves the testing of multiple probe proteins(i.e., “probe protein folds”) spanning the range of a physical propertyof interest (i.e., a “property of interest”) in an appropriate assay.Probe proteins include a limited set of proteins and/or variantsthereof. In some exemplary embodiments, at least one serine protease wastested for one or more benefits. For instance, the change in net chargeof the variant relative to the parent enzyme for two serine proteases,GG36 and FNA were provided. The net charge change for the variants ofGG36 and FNA as described herein, span a relative net charge changerange of −7 to 0 as compared to the respective parent enzymes.

II. Determine Physical Property Optimum

Determining the physical property optimum i.e. determining the optimumof a property of interest, involves identifying a physical propertyoptimum or range thereof for a favorable outcome. In some exemplaryembodiments, the cleaning performance index, herein provided as the BMIPI of FNA and GG36 protease variants was measured. In other embodiments,the expression level herein provided as the TCA PI of the FNA and GG36protease variants was measured. In the present embodiments, whencomparing benefits obtained with proteins having the same fold i.e.serine proteases, a relative was be employed (e.g., net chargedifferential relative to wild-type or parent protein). Alternatively,when comparing benefits obtained with proteins having different folds,e.g. serine proteases and metalloproteases, a common physical propertyscale is employed (e.g., protein charge reported as zeta potential).Probe proteins spanning a wide physical property range are employed, inorder to increase the likelihood of defining an optimum for thatphysical property. Once the optimum value or optimum range for a benefitof interest has been established by assaying the probe proteins, it ispossible to predict both the general direction and magnitude of changelikely to be required for converting an inferior performer (e.g., lyingoutside of the optimal range) to a superior performer (e.g., within theoptimal range).

Use of more than one probe protein series is contemplated to permit theidentification of different physical property optima for a benefit ofinterest. For instance in some embodiments, both changes in net chargeand hydrophobicity relative to that of the parent enzyme of thedetergent proteases GG36 and FNA are tested for cleaning performance ina blood, milk, ink assay. In some instances, the same physical propertyis contemplated to exhibit different optima for different benefits. Forexample, there exists an optimal protease charge for cleaningperformance for FNA (BMI PI), which is distinct from the optimalprotease charge for expression level (TCA PI).

In some embodiments, charge-related physical properties are comparedacross parent and variant proteins in terms of measured zeta potential,net charge, charge density, and/or surface count of ionizable groups. Ingeneral, any method of determining protein charge from titration orelectrophoretic measurements is suitable for comparing different proteinfolds. Comparing different protein variants is done by calculation ofone or more of the above quantities based upon protein primary,secondary and/or tertiary sequence information when available. Typicalbioinformatics tools employed for such purposes include isoelectricpoint calculators using the Henderson-Hesselbach equation (e.g.,European Molecular Biology Laboratory) or Poisson-Boltzmannelectrostatic solvers (e.g., DelPhi, MOE).

III. Provide Variant Proteins Having the Physical Property Optimum

Once an optimum value or range has been determined in the previous step,a plurality of candidate proteins are provided which are constrained forthe physical property of interest. Suitable methods for providingcandidate proteins include, but are not limited to the production ofartificial enzymes variants by recombinant techniques, as well as thepurification of natural enzyme variants (e.g., homologues) bychromatography, the in vitro synthesis of glycosylation orphosphorylation enzyme variants or the in vitro production of enzymeconjugates. Another way to alter the hydrophobicity of a protein viaglycosylation is to generate new glycosylation sites on the surface ofthe enzyme. These variants will be glycosylated in vivo duringexpression.

In some embodiments, hydrophobicity-related physical properties arecompared across protease variants in terms of the overall contributionby the substituted residues to the net change in hydrophobicity of theresulting variant relative to that of the parent enzyme. In someembodiments, the overall hydrophobic contribution is calculated usingone or more of the many amino-acid hydrophobicity scales available inthe literature and known to those in the art, that take into accountprotein primary, secondary and/or tertiary structure information. Ininstances when hydrophobicity-related physical properties are comparedacross different protein folds in terms of measured protein partitioningbetween its native aqueous environment and a hydrophobic phase. Examplesinclude but are not limited to surface tension at the air-water orheptane-water interfaces, as well as contact angle and wettingmeasurements between aqueous and solid substrate-containing phases. Ingeneral, any method suitable for characterizing the partitioning of aprotein between two phases is suitable for use in the present invention,including optical (e.g., ellipsometry, surface plasmon resonance,interferometry, and/or reflectivity), acoustic (e.g., quartz-crystalmicrobalance), fluorescence, spectroscopy (e.g., attenuated totalreflection infrared) or concentration (e.g., enzyme activity)determinations.

Charge and hydrophobicity scales are not independent from each othersince charged residues add hydrophilic character. Thus, rather thansimply choosing one scale over another, some embodiments of the presentinvention employ multiple different scales (e.g., theoretical orexperimentally determined) for identifying physical propertydependencies. References for 23 of the most commonly used hydrophobicityscales include: hydrophobicity (Rao and Argos) calculates membraneburied helix parameter. (Rao and Argos, Biochim. Biophys. Acta869:197-214 [1986]); hydrophobicity (Black and Mould) calculateshydrophobicity of physiological L-alpha amino acids (Black and Mould,Anal. Biochem., 193:72-82 [1991]); hydrophobicity (Bull and Breese)calculates hydrophobicity (free energy of transfer to surface inkcal/mole) (Bull and Breese, Arch. Biochem. Biophys. 161:665-670[1974]); hydrophobicity (Chothia) calculates proportion of residues 95%buried (in 12 proteins) (Chothia, J. Mol. Biol., 105:1-14 [1976]);hydrophobicity (Kyte and Doolittle) calculates hydropathicity (Kyte andDoolittle, J. Mol. Biol., 157:105-132 [1982]); hydrophobicity (Eisenberget al.) calculates normalized consensus hydrophobicity scale (Eisenberget al., J. Mol. Biol. 179:125-142 [1984]); hydrophobicity (Fauchere andPliska) calculates hydrophobicity scale (pi-r) (Fauchere and Pliska,Eur. J. Med. Chem., 18:369-375 [1983]); hydrophobicity (Guy) calculateshydrophobicity scale based on free energy of transfer (kcal/mole) (Guy,Biophys J., 47:61-70 [1985]); hydrophobicity (Janin) calculates freeenergy of transfer from inside to outside of a globular protein (Janin,Nature 277:491-492 [1979]); hydrophobicity (Abraham and Leo) calculateshydrophobicity (delta G1/2cal) (Abraham and Leo, Proteins: Structure,Function and Genetics 2:130-152 [1987]); hydrophobicity (Manavalan etal.) calculates average surrounding hydrophobicity (Manavalan et al.,Nature 275:673-674 [1978]); Hydrophobicity (Miyazawa et al.) calculateshydrophobicity scale (contact energy derived from 3D data) (Miyazawa etal., Macromolecules 18:534-552 [1985]); hydrophobicity (Aboderin)calculates mobilities of amino acids on chromatography paper (RF)(Aboderin, Int. J. Biochem., 2:537-544 [1971]); hydrophobicity HPLC(Parker et al.) calculates hydrophilicity scale derived from HPLCpeptide retention times (Parker et al., Biochem., 25:5425-5431 [1986]);Hphob. HPLC pH3.4 calculates hydrophobicity indices at ph 3.4 determinedby HPLC (Cowan and Whittaker, Peptide Res., 3:75-80 [1990]); Hphob. HPLCpH7.5 calculates hydrophobicity indices at ph 7.5 determined by HPLC(Cowan and Whittaker, Peptide Res., 3:75-80 [1990]); hydrophobicity(Rose et al.) (AA) calculates the mean fractional area loss (f) [averagearea buried/standard state area] (Rose et al., Science 229:834-838[1985)); and hydrophobicity (Roseman) calculates hydrophobicity scale(pi-r) (Roseman, J. Mol. Biol., 200:513-522 [1988)).

Other physical properties that are compared across proteases andvariants thereof within the same protein fold or across differentprotein folds include but are not limited to solubility-related physicalproperties, size-related physical properties, and protein meltingtemperatures. Solubility-related physical properties are compared acrossdifferent protein folds in terms of both charge and hydrophobicityscales previously described. In general, any thermodynamic or kineticquantity characterizing protein-protein versus protein-solventinteractions is suitable for use with the methods of the presentinvention. For instance second virial coefficient (See, Wilson, ActaCrystallographica, D50:361-365 [1994]), chi parameter, osmotic pressure,and activity or fugacity coefficients reflecting deviations from idealmixing behavior find use (See e.g., Reid et al., “The Properties ofGases and Liquids”, 4^(th) Ed. McGraw-Hill, [1987]). Size-relatedphysical properties are compared across different protein folds usingany experimental means suitable for determining protein or polymerdimensions. Size is inferred from molecular weight using commonlyavailable correlations between protein or polymer conformation (coil,globular, branched), their molecular weight and hydrodynamic or gyrationradius. Suitable techniques for size or molecular weight determinationinclude, but are not limited to static and dynamic light scattering, gelelectrophoresis, mass spectroscopy and chromatography. Alternatively,size is readily estimated from knowledge of the experimentallydetermined protein crystal structures or structural homology models.

Protein melting temperatures (T_(m)) are typically determined throughmonitoring of a physical reporter property across a temperature scan.Suitable methods include, but are not limited to differential scanningcalorimetry, circular dichroism, dynamic light scattering, andUV-visible spectroscopy.

Applications for Serine Protease Enzymes

The combinable mutations created to generate the protease variants arecontemplated to serve to enhance the performance index e.g. locate theperformance optimum, for at least one desired enzyme property in avariety of applications. The location of the performance/propertyoptimum is largely influenced by medium utilized (e.g., detergentformulation, pH, ionic strength, etc), as well as the net charge andcharge distribution of amino acid residues of the enzyme of interest.Thus, an optimal enzyme is contemplated to exist for differentformulations of varying pH, ionic strength, surfactant type and ratio,builders and chelators, all of which affect electrostatic phenomena. Theuse of enzyme blends, in which each member of the blend possesses adifferent charge optimum, is contemplated for the production offormulations suitable for a wide range of conditions (e.g., proteases indetergent formulations sold in different geographies or locales havingdifferences in water hardness). The use of enzyme blends, in which eachmember of the blend excels in the cleaning of a different stain, is alsocontemplated for the production of formulations suitable for cleaning awide variety of stains. Additionally, the use of enzyme blends, in whicheach member of the blend possesses a different charge optimum, iscontemplated in cases where the enzyme substrate itself undergoes chargechanges during enzyme reaction

Although described herein in relationship to proteases and blood, milkand ink stains, it is contemplated that the protease variants of thepresent invention are optimized for any enzyme-substrate interaction inany a variety of reaction media (i.e. conditions) dictated by theapplication e.g. cleaning applications.

As described in greater detail herein, the variant proteases of thepresent invention have important characteristics that make them verysuitable for certain applications. For example, in some preferredembodiments, the variant proteases of the present invention have alteredcharge and/or hydrophobicity as compared to some currently usedproteases. Thus, these proteases find particular use in cleaningcompositions. Indeed, under certain wash conditions, the presentproteases exhibit comparative or enhanced expression and/or stainremoval activity as compared with currently used subtilisin proteases.Thus, it is contemplated that the cleaning and/or enzyme compositions ofthe present invention will be provided in a variety of cleaningcompositions. Thus, the present proteases find use in various cleaningcompositions, as well as animal feed applications, leather processing(e.g., bating), protein hydrolysis, and in textile uses. The identifiedproteases also find use in personal care applications.

Indeed, the protease variants of the present invention find use in anumber of industrial applications, in particular within the cleaning,disinfecting, animal feed, and textile/leather industries. In someembodiments, the protease(s) of the present invention are combined withdetergents, builders, bleaching agents and other conventionalingredients to produce a variety of novel cleaning compositions usefulin the laundry and other cleaning arts such as, for example, laundrydetergents (both powdered and liquid), laundry pre-soaks, all fabricbleaches, automatic dishwashing detergents (both liquid and powdered),household cleaners, particularly bar and liquid soap applications, anddrain openers. In addition, the variant proteases find use in thecleaning of contact lenses, as well as other items, by contacting suchmaterials with an aqueous solution of the cleaning composition. Inaddition these variant proteases can be used, for example in peptidehydrolysis, waste treatment, textile applications, medical devicecleaning, biofilm removal and as fusion-cleavage enzymes in proteinproduction, etc. The composition of these products is not critical tothe present invention, as long as the variant protease(s) maintain theirfunction in the setting used. In some embodiments, the compositions arereadily prepared by combining a cleaning effective amount of theprotease variant or an enzyme composition comprising the variantprotease enzyme preparation with the conventional components of suchcompositions in their art recognized amounts.

Cleaning Compositions

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources. Enzyme components weights are based on total active protein.All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated. In the exemplified detergentcompositions, the enzymes levels are expressed by pure enzyme by weightof the total composition and unless otherwise specified, the detergentingredients are expressed by weight of the total compositions.

As indicated herein, in some embodiments, the cleaning compositions ofthe present invention further comprise adjunct materials including, butnot limited to, surfactants, builders, bleaches, bleach activators,bleach catalysts, other enzymes, enzyme stabilizing systems, chelants,optical brighteners, soil release polymers, dye transfer agents,dispersants, suds suppressors, dyes, perfumes, colorants, filler salts,hydrotropes, photoactivators, fluorescers, fabric conditioners,hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkageagents, anti-wrinkle agents, germicides, fungicides, color speckles,silvercare, anti-tarnish and/or anti-corrosion agents, alkalinitysources, solubilizing agents, carriers, processing aids, pigments, andpH control agents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458,5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, allof which are incorporated herein by reference). Embodiments of specificcleaning composition materials are exemplified in detail below. Inembodiments in which the cleaning adjunct materials are not compatiblewith the variant proteases of the present invention in the cleaningcompositions, then suitable methods of keeping the cleaning adjunctmaterials and the protease(s) separated (i.e., not in contact with eachother) until combination of the two components is appropriate are used.Such separation methods include any suitable method known in the art(e.g., gelcaps, encapsulation, tablets, physical separation, etc.).

The cleaning compositions of the present invention are advantageouslyemployed for example, in laundry applications, hard surface cleaning,dishwashing applications, as well as cosmetic applications such asdentures, teeth, hair and skin. In addition, due to the uniqueadvantages of increased effectiveness in lower temperature solutions,the enzymes of the present invention are ideally suited for laundryapplications. Furthermore, the enzymes of the present invention find usein granular and liquid compositions.

The variant proteases of the present invention also find use cleaningadditive products. In some embodiments, low temperature solutioncleaning applications find use. In some embodiments, the presentinvention provides cleaning additive products including at least oneenzyme of the present invention is ideally suited for inclusion in awash process when additional bleaching effectiveness is desired. Suchinstances include, but are not limited to low temperature solutioncleaning applications. In some embodiments, the additive product is inits simplest form, one or more proteases. In some embodiments, theadditive is packaged in dosage form for addition to a cleaning process.In some embodiments, the additive is packaged in dosage form foraddition to a cleaning process where a source of peroxygen is employedand increased bleaching effectiveness is desired. Any suitable singledosage unit form finds use with the present invention, including but notlimited to pills, tablets, gelcaps, or other single dosage units such aspre-measured powders or liquids. In some embodiments, filler(s) orcarrier material(s) are included to increase the volume of suchcompositions. Suitable filler or carrier materials include, but are notlimited to, various salts of sulfate, carbonate and silicate as well astalc, clay and the like. Suitable filler or carrier materials for liquidcompositions include, but are not limited to water or low molecularweight primary and secondary alcohols including polyols and diols.Examples of such alcohols include, but are not limited to, methanol,ethanol, propanol and isopropanol. In some embodiments, the compositionscontain from about 5% to about 90% of such materials. Acidic fillersfind use to reduce pH. Alternatively, in some embodiments, the cleaningadditive includes adjunct ingredients, as more fully described below.

The present cleaning compositions and cleaning additives require aneffective amount of at least one of the protease variants providedherein, alone or in combination with other proteases and/or additionalenzymes. The required level of enzyme is achieved by the addition of oneor more protease variants of the present invention. Typically thepresent cleaning compositions will comprise at least about 0.0001 weightpercent, from about 0.0001 to about 10, from about 0.001 to about 1, oreven from about 0.01 to about 0.1 weight percent of at least one of thevariant proteases of the present invention.

The cleaning compositions herein are typically formulated such that,during use in aqueous cleaning operations, the wash water will have a pHof from about 5.0 to about 11.5 or even from about 7.5 to about 10.5.Liquid product formulations are typically formulated to have a neat pHfrom about 3.0 to about 9.0 or even from about 3 to about 5. Granularlaundry products are typically formulated to have a pH from about 9 toabout 11. Techniques for controlling pH at recommended usage levelsinclude the use of buffers, alkalis, acids, etc., and are well known tothose skilled in the art.

Suitable low pH cleaning compositions typically have a neat pH of fromabout 3 to about 5, and are typically free of surfactants that hydrolyzein such a pH environment. Such surfactants include sodium alkyl sulfatesurfactants that comprise at least one ethylene oxide moiety or evenfrom about 1 to about 16 moles of ethylene oxide. Such cleaningcompositions typically comprise a sufficient amount of a pH modifier,such as sodium hydroxide, monoethanolamine or hydrochloric acid, toprovide such cleaning composition with a neat pH of from about 3 toabout 5. Such compositions typically comprise at least one acid stableenzyme. In some embodiments, the compositions are liquids, while inother embodiments, they are solids. The pH of such liquid compositionsis typically measured as a neat pH. The pH of such solid compositions ismeasured as a 10% solids solution of said composition wherein thesolvent is distilled water. In these embodiments, all pH measurementsare taken at 20° C., unless otherwise indicated.

In some embodiments, when the variant protease(s) is/are employed in agranular composition or liquid, it is desirable for the variant proteaseto be in the form of an encapsulated particle to protect the variantprotease from other components of the granular composition duringstorage. In addition, encapsulation is also a means of controlling theavailability of the variant protease during the cleaning process. Insome embodiments, encapsulation enhances the performance of the variantprotease(s) and/or additional enzymes. In this regard, the variantproteases of the present invention are encapsulated with any suitableencapsulating material known in the art. In some embodiments, theencapsulating material typically encapsulates at least part of thecatalyst for the variant protease(s) of the present invention.Typically, the encapsulating material is water-soluble and/orwater-dispersible. In some embodiments, the encapsulating material has aglass transition temperature (Tg) of 0° C. or higher. Glass transitiontemperature is described in more detail in WO 97/11151. Theencapsulating material is typically selected from consisting ofcarbohydrates, natural or synthetic gums, chitin, chitosan, celluloseand cellulose derivatives, silicates, phosphates, borates, polyvinylalcohol, polyethylene glycol, paraffin waxes, and combinations thereof.When the encapsulating material is a carbohydrate, it is typicallyselected from monosaccharides, oligosaccharides, polysaccharides, andcombinations thereof. In some typical embodiments, the encapsulatingmaterial is a starch (See e.g., EP 0 922 499; U.S. Pat. Nos. 4,977,252;5,354,559, and 5,935,826). In some embodiments, the encapsulatingmaterial is a microsphere made from plastic such as thermoplastics,acrylonitrile, methacrylonitrile, polyacrylonitrile,polymethacrylonitrile and mixtures thereof; commercially availablemicrospheres that find use include, but are not limited to thosesupplied by EXPANCEL® (Stockviksverken, Sweden), and PM 6545, PM 6550,PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQCorp., Valley Forge, Pa.).

As described herein, the variant proteases of the present invention findparticular use in the cleaning industry, including, but not limited tolaundry and dish detergents. These applications place enzymes undervarious environmental stresses. The variant proteases of the presentinvention provide advantages over many currently used enzymes, due totheir stability under various conditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which proteases involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, European detergentstypically have about 4500-5000 ppm of detergent components in the washwater, while Japanese detergents typically have approximately 667 ppm ofdetergent components in the wash water. In North America, particularlythe United States, detergents typically have about 975 ppm of detergentcomponents present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan is typically between about 10 and about30° C. (e.g., about 20° C.), whereas the temperature of wash water inEurope is typically between about 30 and about 60° C. (e.g., about 40°C.). However, in the interest of saving energy, many consumers areswitching to using cold water washing. In addition, in some furtherregions, cold water is typically used for laundry, as well as dishwashing applications. In some embodiments, the “cold water washing” ofthe present invention utilizes washing at temperatures from about 10° C.to about 40° C., or from about 10° C. to about 30° C., or from about 15°C. to about 25° C., as well as all other combinations within the rangeof about 10° C. to about 40° C. As a further example, differentgeographies typically have different water hardness. Water hardness isusually described in terms of the grains per gallon mixed Ca²⁺/Mg²⁺.Hardness is a measure of the amount of calcium (Ca²⁺) and magnesium(Mg²⁺) in the water. Most water in the United States is hard, but thedegree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181ppm) water has 60 to 181 parts per million (parts per million convertedto grains per U.S. gallon is ppm # divided by 17.1 equals grains pergallon) of hardness minerals.

Water Grains per gallon Parts per million Soft less than 1.0 less than17 Slightly hard 1.0 to 3.5  17 to 60 Moderately hard 3.5 to 7.0  60 to120 Hard 7.0 to 10.5 120 to 180 Very hard greater than 10.5 greater than180

European water hardness is typically greater than about 10.5 (forexample about 10.5 to about 20.0) grains per gallon mixed Ca²⁺/Mg²⁺,(e.g., about 15 grains per gallon mixed Ca²⁺/Mg²⁺). North American waterhardness is typically greater than Japanese water hardness, but lessthan European water hardness. For example, North American water hardnesscan be between about 3 to about 10 grains, about 3 to about 8 grains orabout 6 grains. Japanese water hardness is typically lower than NorthAmerican water hardness, usually less than about 4, for example about 3grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present invention provides variantproteases that show surprising wash performance in at least one set ofwash conditions (e.g., water temperature, water hardness, and/ordetergent concentration). In some embodiments, the variant proteases ofthe present invention are comparable in wash performance to othersubtilisin proteases. In some embodiments, the variant proteases of thepresent invention exhibit enhanced wash performance as compared tosubtilisin proteases currently commercially available. Thus, in somepreferred embodiments of the present invention, the variant proteasesprovided herein exhibit enhanced oxidative stability, enhanced thermalstability, enhanced cleaning capabilities under various conditions,and/or enhanced chelator stability. In addition, the variant proteasesof the present invention find use in cleaning compositions that do notinclude detergents, again either alone or in combination with buildersand stabilizers.

In some embodiments of the present invention, the cleaning compositionscomprise at least one variant protease of the present invention at alevel from about 0.00001% to about 10% by weight of the composition andthe balance (e.g., about 99.999% to about 90.0%) comprising cleaningadjunct materials by weight of composition. In other aspects of thepresent invention, the cleaning compositions of the present inventioncomprises at least one variant protease at a level of about 0.0001% toabout 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% by weight of the composition and the balance of thecleaning composition (e.g., about 99.9999% to about 90.0%, about 99.999%to about 98%, about 99.995% to about 99.5% by weight) comprisingcleaning adjunct materials.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more additional detergent enzymes, which providecleaning performance and/or fabric care and/or dishwashing benefits.Examples of suitable enzymes include, but are not limited to,hemicellulases, cellulases, peroxidases, proteases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, pectate lyases,mannanases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, acombination of enzymes is used (i.e., a “cocktail”) comprisingconventional applicable enzymes like protease, lipase, cutinase and/orcellulase in conjunction with amylase is used.

In addition to the protease variants provided herein, any other suitableprotease finds use in the compositions of the present invention.Suitable proteases include those of animal, vegetable or microbialorigin. In some particularly preferred embodiments, microbial proteasesare used. In some embodiments, chemically or genetically modifiedmutants are included. In some embodiments, the protease is a serineprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases include subtilisins, especiallythose derived from Bacillus (e.g., subtilisin, lentus,amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147and subtilisin 168). Additional examples include those mutant proteasesdescribed in U.S. Pat. No. RE 34,606, U.S. Pat. Nos. 5,955,340,5,700,676, 6,312,936, and 6,482,628, all of which are incorporatedherein by reference. Additional protease examples include, but are notlimited to trypsin (e.g., of porcine or bovine origin), and the Fusariumprotease described in WO 89/06270. In some embodiments, commerciallyavailable protease enzymes that find use in the present inventioninclude, but are not limited to MAXATASE®, MAXACAL™, MAXAPEM™,OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® PROPERASE®, EXCELLASE™,PURAFAST™, and PURAFECT® OXP (Genencor); ALCALASE®, SAVINASE®, PRIMASE®,DURAZYM™, POLARZYME®, OVOZYME®, LIQUANASE®, KANNASE®, NEUTRASE®, RELASE®and ESPERASE® (Novozymes); and BLAP™ (Henkel Kommanditgesellschaft aufAktien, Duesseldorf, Germany. Various proteases are described inWO95/23221, WO 92/21760, U.S. Pat. Appln. Publ. No. 2008/0090747, andU.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, US RE 34,606,U.S. Pat. Nos. 5,955,340, 5,700,676, 6,312,936, and 6,482,628, andvarious other patents. In some embodiments, metalloproteases find use inthe present invention, including, but not limited to the neutralmetalloprotease described in WO 07/044993.

In addition, any suitable lipase finds use in the present invention.Suitable lipases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants areencompassed by the present invention. Examples of useful lipases includeHumicola lanuginosa lipase (See e.g., EP 258 068, and EP 305 216),Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such asC. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g.,EP 214 761), Pseudomonas lipases such as P. alcaligenes lipase and P.pseudoalcaligenes lipase (See e.g., EP 218 272), P. cepacia lipase (Seee.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P.fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartoiset al., Biochem. Biophys. Acta 1131:253-260 [1993]); B.stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumiluslipase [See e.g., WO 91/16422]).

Furthermore, a number of cloned lipases find use in some embodiments ofthe present invention, including but not limited to Penicilliumcamembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]),Geotricum candidum lipase (See, Schimada et al., J. Biochem.,106:383-388 [1989]), and various Rhizopus lipases such as R. delemarlipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase(Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R.oryzae lipase.

Other types of lipolytic enzymes such as cutinases also find use in someembodiments of the present invention, including but not limited to thecutinase derived from Pseudomonas mendocina (See, WO 88/09367), and thecutinase derived from Fusarium solani pisi (See, WO 90/09446).

Additional suitable lipases include commercially available lipases suchas M1 LIPASE™ LUMA FAST™, and LIPOMAX™ (Genencor); LIPOLASE® andLIPOLASE® ULTRA (Novozymes); and LIPASE P™ “Amano” (Amano PharmaceuticalCo. Ltd., Japan).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise lipases at a level from about0.00001% to about 10% of additional lipase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In other aspects of the present invention, the cleaning compositions ofthe present invention also comprise lipases at a level of about 0.0001%to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% lipase by weight of the composition.

In some embodiments of the present invention, any suitable amylase findsuse in the present invention. In some embodiments, any amylase (e.g.,alpha and/or beta) suitable for use in alkaline solutions also find use.Suitable amylases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants are includedin some embodiments. Amylases that find use in the present invention,include, but are not limited to α-amylases obtained from B.licheniformis (See e.g., GB 1,296,839). Commercially available amylasesthat find use in the present invention include, but are not limited toDURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYMEULTRA®, NATALASE®, and BAN™ (Novozymes), as well as POWERASE™, RAPIDASE®and MAXAMYL® P (Genencor).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise amylases at a level from about0.00001% to about 10% of additional amylase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In other aspects of the present invention, the cleaning compositions ofthe present invention also comprise amylases at a level of about 0.0001%to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% amylase by weight of the composition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present invention. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited toHumicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).Especially suitable cellulases are the cellulases having color carebenefits (See e.g., EP 0 495 257). Commercially available cellulasesthat find use in the present include, but are not limited to CELLUZYME®,CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation). In someembodiments, cellulases are incorporated as portions or fragments ofmature wild-type or variant cellulases, wherein a portion of theN-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276). In someembodiments, the cleaning compositions of the present invention furthercomprise cellulases at a level from about 0.00001% to about 10% ofadditional cellulase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In other aspects ofthe present invention, the cleaning compositions of the presentinvention also comprise cellulases at a level of about 0.0001% to about10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% toabout 0.5% cellulase by weight of the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present invention. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present invention (See e.g.,U.S. Pat. Nos. 6,566,114, 6,602,842, and 6,440,991, all of which areincorporated herein by reference). In some embodiments, the cleaningcompositions of the present invention further comprise mannanases at alevel from about 0.00001% to about 10% of additional mannanase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In other aspects of the present invention, thecleaning compositions of the present invention also comprise mannanasesat a level of about 0.0001% to about 10%, about 0.001% to about 5%,about 0.001% to about 2%, about 0.005% to about 0.5% mannanase by weightof the composition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present invention. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO 94/12621 and WO95/01426). Suitable peroxidases/oxidases include, but are not limited tothose of plant, bacterial or fungal origin. Chemically or geneticallymodified mutants are included in some embodiments. In some embodiments,the cleaning compositions of the present invention further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001% toabout 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In other aspects of the present invention, the cleaningcompositions of the present invention also comprise, peroxidase and/oroxidase enzymes at a level of about 0.0001% to about 10%, about 0.001%to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%peroxidase and/or oxidase enzymes by weight of the composition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO 05/056782). In addition, in someparticularly preferred embodiments, mixtures of the above mentionedenzymes are encompassed herein, in particular one or more additionalprotease, amylase, lipase, mannanase, and/or at least one cellulase.Indeed, it is contemplated that various mixtures of these enzymes willfind use in the present invention. It is also contemplated that thevarying levels of the variant protease(s) and one or more additionalenzymes may both independently range to about 10%, the balance of thecleaning composition being cleaning adjunct materials. The specificselection of cleaning adjunct materials are readily made by consideringthe surface, item, or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use (e.g., through thewash detergent use).

Examples of suitable cleaning adjunct materials include, but are notlimited to, surfactants, builders, bleaches, bleach activators, bleachcatalysts, other enzymes, enzyme stabilizing systems, chelants, opticalbrighteners, soil release polymers, dye transfer agents, dye transferinhibiting agents, catalytic materials, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracis, polymeric dispersing agents, claysoil removal agents, structure elasticizing agents, dispersants, sudssuppressors, dyes, perfumes, colorants, filler salts, hydrotropes,photoactivators, fluorescers, fabric conditioners, fabric softeners,carriers, hydrotropes, processing aids, solvents, pigments, hydrolyzablesurfactants, preservatives, anti-oxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, and pH controlagents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464,5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of whichare incorporated herein by reference). Embodiments of specific cleaningcomposition materials are exemplified in detail below. In embodiments inwhich the cleaning adjunct materials are not compatible with the variantproteases of the present invention in the cleaning compositions, thensuitable methods of keeping the cleaning adjunct materials and theprotease(s) separated (i.e., not in contact with each other) untilcombination of the two components is appropriate are used. Suchseparation methods include any suitable method known in the art (e.g.,gelcaps, encapsulation, tablets, physical separation, etc.).

In some preferred embodiments, an effective amount of one or morevariant protease(s) provided herein are included in compositions usefulfor cleaning a variety of surfaces in need of proteinaceous stainremoval. Such cleaning compositions include cleaning compositions forsuch applications as cleaning hard surfaces, fabrics, and dishes.Indeed, in some embodiments, the present invention provides fabriccleaning compositions, while in other embodiments, the present inventionprovides non-fabric cleaning compositions. Notably, the presentinvention also provides cleaning compositions suitable for personalcare, including oral care (including dentrifices, toothpastes,mouthwashes, etc., as well as denture cleaning compositions), skin, andhair cleaning compositions. It is intended that the present inventionencompass detergent compositions in any form (i.e., liquid, granular,bar, semi-solid, gels, emulsions, tablets, capsules, etc.).

By way of example, several cleaning compositions wherein the variantproteases of the present invention find use are described in greaterdetail below. In some embodiments in which the cleaning compositions ofthe present invention are formulated as compositions suitable for use inlaundry machine washing method(s), the compositions of the presentinvention preferably contain at least one surfactant and at least onebuilder compound, as well as one or more cleaning adjunct materialspreferably selected from organic polymeric compounds, bleaching agents,additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. In some embodiments, laundry compositions also containsoftening agents (i.e., as additional cleaning adjunct materials). Thecompositions of the present invention also find use detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement and/or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process. Insome embodiments, the density of the laundry detergent compositionsherein ranges from about 400 to about 1200 g/liter, while in otherembodiments, it ranges from about 500 to about 950 g/liter ofcomposition measured at 20° C.

In embodiments formulated as compositions for use in manual dishwashingmethods, the compositions of the invention preferably contain at leastone surfactant and preferably at least one additional cleaning adjunctmaterial selected from organic polymeric compounds, suds enhancingagents, group II metal ions, solvents, hydrotropes and additionalenzymes.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458 find use with the variant proteasesof the present invention. Thus, in some embodiments, the compositionscomprising at least one variant protease of the present invention is acompact granular fabric cleaning composition, while in otherembodiments, the composition is a granular fabric cleaning compositionuseful in the laundering of colored fabrics, in further embodiments, thecomposition is a granular fabric cleaning composition which providessoftening through the wash capacity, in additional embodiments, thecomposition is a heavy duty liquid fabric cleaning composition. In someembodiments, the compositions comprising at least one variant proteaseof the present invention are fabric cleaning compositions such as thosedescribed in U.S. Pat. Nos. 6,610,642 and 6,376,450. In addition, thevariant proteases of the present invention find use in granular laundrydetergent compositions of particular utility under European or Japanesewashing conditions (See e.g., U.S. Pat. No. 6,610,642).

In some alternative embodiments, the present invention provides hardsurface cleaning compositions comprising at least one variant proteaseprovided herein. Thus, in some embodiments, the compositions comprisingat least one variant protease of the present invention is a hard surfacecleaning composition such as those described in U.S. Pat. Nos.6,610,642, 6,376,450, and 6,376,450.

In yet further embodiments, the present invention provides dishwashingcompositions comprising at least one variant protease provided herein.Thus, in some embodiments, the compositions comprising at least onevariant protease of the present invention is a hard surface cleaningcomposition such as those in U.S. Pat. Nos. 6,610,642 and 6,376,450. Insome still further embodiments, the present invention providesdishwashing compositions comprising at least one variant proteaseprovided herein. In some further embodiments, the compositionscomprising at least one variant protease of the present inventioncomprise oral care compositions such as those in U.S. Pat. Nos.6,376,450, and 6,376,450. The formulations and descriptions of thecompounds and cleaning adjunct materials contained in the aforementionedU.S. Pat. Nos. 6,376,450, 6,605,458, 6,605,458, and 6,610,642, find usewith the variant proteases provided herein.

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in U.S. Pat. Nos.5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. When a low pH cleaning composition is desired, the pH of suchcomposition is adjusted via the addition of a material such asmonoethanolamine or an acidic material such as HCl.

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant cleaning compositions. In some embodiments, theseadjuncts are incorporated for example, to assist or enhance cleaningperformance, for treatment of the substrate to be cleaned, or to modifythe aesthetics of the cleaning composition as is the case with perfumes,colorants, dyes or the like. It is understood that such adjuncts are inaddition to the variant proteases of the present invention. The precisenature of these additional components, and levels of incorporationthereof, will depend on the physical form of the composition and thenature of the cleaning operation for which it is to be used. Suitableadjunct materials include, but are not limited to, surfactants,builders, chelating agents, dye transfer inhibiting agents, depositionaids, dispersants, additional enzymes, and enzyme stabilizers, catalyticmaterials, bleach activators, bleach boosters, hydrogen peroxide,sources of hydrogen peroxide, preformed peracids, polymeric dispersingagents, clay soil removal/anti-redeposition agents, brighteners, sudssuppressors, dyes, perfumes, structure elasticizing agents, fabricsofteners, carriers, hydrotropes, processing aids and/or pigments. Inaddition to the disclosure below, suitable examples of such otheradjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282,6,306,812, and 6,326,348, incorporated by reference. The aforementionedadjunct ingredients may constitute the balance of the cleaningcompositions of the present invention.

In some embodiments, the cleaning compositions according to the presentinvention comprise at least one surfactant and/or a surfactant systemwherein the surfactant is selected from nonionic surfactants, anionicsurfactants, cationic surfactants, ampholytic surfactants, zwitterionicsurfactants, semi-polar nonionic surfactants and mixtures thereof. Insome low pH cleaning composition embodiments (e.g., compositions havinga neat pH of from about 3 to about 5), the composition typically doesnot contain alkyl ethoxylated sulfate, as it is believed that suchsurfactant may be hydrolyzed by such compositions the acidic contents.In some embodiments, the surfactant is present at a level of from about0.1% to about 60%, while in alternative embodiments the level is fromabout 1% to about 50%, while in still further embodiments the level isfrom about 5% to about 40%, by weight of the cleaning composition.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent builders or builder systems. In someembodiments incorporating at least one builder, the cleaningcompositions comprise at least about 1%, from about 3% to about 60% oreven from about 5% to about 40% builder by weight of the cleaningcomposition. Builders include, but are not limited to, the alkali metal,ammonium and alkanolammonium salts of polyphosphates, alkali metalsilicates, alkaline earth and alkali metal carbonates, aluminosilicates,polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof. Indeed, it is contemplated that any suitablebuilder will find use in various embodiments of the present invention.

In some embodiments, the builders form water-soluble hardness ioncomplexes (e.g., sequestering builders), such as citrates andpolyphosphates (e.g., sodium tripolyphosphate and sodiumtripolyphosphate hexahydrate, potassium tripolyphosphate, and mixedsodium and potassium tripolyphosphate, etc.). It is contemplated thatany suitable builder will find use in the present invention, includingthose known in the art (See e.g., EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioncontain at least one chelating agent. Suitable chelating agents include,but are not limited to copper, iron and/or manganese chelating agentsand mixtures thereof. In embodiments in which at least one chelatingagent is used, the cleaning compositions of the present inventioncomprise from about 0.1% to about 15% or even from about 3.0% to about10% chelating agent by weight of the subject cleaning composition.

In some still further embodiments, the cleaning compositions providedherein contain at least one deposition aid. Suitable deposition aidsinclude, but are not limited to, polyethylene glycol, polypropyleneglycol, polycarboxylate, soil release polymers such as polytelephthalicacid, clays such as kaolinite, montmorillonite, atapulgite, illite,bentonite, halloysite, and mixtures thereof.

As indicated herein, in some embodiments, anti-redeposition agents finduse in some embodiments of the present invention. In some preferredembodiments, non-ionic surfactants find use. For example, in automaticdishwashing embodiments, non-ionic surfactants find use for surfacemodification purposes, in particular for sheeting, to avoid filming andspotting and to improve shine. These non-ionic surfactants also find usein preventing the re-deposition of soils. In some preferred embodiments,the anti-redeposition agent is a non-ionic surfactant as known in theart (See e.g., EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioninclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. In embodiments in which atleast one dye transfer inhibiting agent is used, the cleaningcompositions of the present invention comprise from about 0.0001% toabout 10%, from about 0.01% to about 5%, or even from about 0.1% toabout 3% by weight of the cleaning composition.

In some embodiments, silicates are included within the compositions ofthe present invention. In some such embodiments, sodium silicates (e.g.,sodium disilicate, sodium metasilicate, and crystalline phyllosilicates)find use. In some embodiments, silicates are present at a level of fromabout 1% to about 20%. In some preferred embodiments, silicates arepresent at a level of from about 5% to about 15% by weight of thecomposition.

In some still additional embodiments, the cleaning compositions of thepresent invention also contain dispersants. Suitable water-solubleorganic materials include, but are not limited to the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

In some further embodiments, the enzymes used in the cleaningcompositions are stabilized any suitable technique. In some embodiments,the enzymes employed herein are stabilized by the presence ofwater-soluble sources of calcium and/or magnesium ions in the finishedcompositions that provide such ions to the enzymes. In some embodiments,the enzyme stabilizers include oligosaccharides, polysaccharides, andinorganic divalent metal salts, including alkaline earth metals, such ascalcium salts. It is contemplated that various techniques for enzymestabilization will find use in the present invention. For example, insome embodiments, the enzymes employed herein are stabilized by thepresence of water-soluble sources of zinc (II), calcium (II) and/ormagnesium (II) ions in the finished compositions that provide such ionsto the enzymes, as well as other metal ions (e.g., barium (II), scandium(II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II),copper (II), nickel (II), and oxovanadium (IV). Chlorides and sulfatesalso find use in some embodiments of the present invention. Examples ofsuitable oligosaccharides and polysaccharides (e.g., dextrins) are knownin the art (See e.g., WO 07/145964). In some embodiments, reversibleprotease inhibitors also find use, such as boron-containing compounds(e.g., borate, 4-formyl phenyl boronic acid) and/or a tripeptidealdehyde find use to further improve stability, as desired.

In some embodiments, bleaches, bleach activators and/or bleach catalystsare present in the compositions of the present invention. In someembodiments, the cleaning compositions of the present invention compriseinorganic and/or organic bleaching compound(s). Inorganic bleachesinclude, but are not limited to perhydrate salts (e.g., perborate,percarbonate, perphosphate, persulfate, and persilicate salts). In someembodiments, inorganic perhydrate salts are alkali metal salts. In someembodiments, inorganic perhydrate salts are included as the crystallinesolid, without additional protection, although in some otherembodiments, the salt is coated. Any suitable salt known in the artfinds use in the present invention (See e.g., EP 2 100 949).

In some embodiments, bleach activators are used in the compositions ofthe present invention. Bleach activators are typically organic peracidprecursors that enhance the bleaching action in the course of cleaningat temperatures of 60° C. and below. Bleach activators suitable for useherein include compounds which, under perhydrolysis conditions, givealiphatic peroxycarboxylic acids having preferably from about 1 to about10 carbon atoms, in particular from about 2 to about 4 carbon atoms,and/or optionally substituted perbenzoic acid. Additional bleachactivators are known in the art and find use in the present invention(See e.g., EP 2 100 949).

In addition, in some embodiments and as further described herein, thecleaning compositions of the present invention further comprise at leastone bleach catalyst. In some embodiments, the manganesetriazacyclononane and related complexes find use, as well as cobalt,copper, manganese, and iron complexes. Additional bleach catalysts finduse in the present invention (See e.g., U.S. Pat. Nos. 4,246,612,5,227,084, 4,810,410, WO 99/06521, and EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioncontain one or more catalytic metal complexes. In some embodiments, ametal-containing bleach catalyst finds use. In some preferredembodiments, the metal bleach catalyst comprises a catalyst systemcomprising a transition metal cation of defined bleach catalyticactivity, (e.g., copper, iron, titanium, ruthenium, tungsten,molybdenum, or manganese cations), an auxiliary metal cation havinglittle or no bleach catalytic activity (e.g., zinc or aluminum cations),and a sequestrate having defined stability constants for the catalyticand auxiliary metal cations, particularly ethylenediaminetetraaceticacid, ethylenediaminetetra (methylenephosphonic acid) and water-solublesalts thereof are used (See e.g., U.S. Pat. No. 4,430,243). In someembodiments, the cleaning compositions of the present invention arecatalyzed by means of a manganese compound. Such compounds and levels ofuse are well known in the art (See e.g., U.S. Pat. No. 5,576,282). Inadditional embodiments, cobalt bleach catalysts find use in the cleaningcompositions of the present invention. Various cobalt bleach catalystsare known in the art (See e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967)and are readily prepared by known procedures.

In additional embodiments, the cleaning compositions of the presentinvention include a transition metal complex of a macropolycyclic rigidligand (MRL). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present invention are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some preferred embodiments, provide from about0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of theMRL in the wash liquor.

Preferred transition-metals in the instant transition-metal bleachcatalyst include, but are not limited to manganese, iron and chromium.Preferred MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO 2000/32601, and U.S. Pat. No. 6,225,464).

In some embodiments, the cleaning compositions of the present inventioncomprise metal care agents. Metal care agents find use in preventingand/or reducing the tarnishing, corrosion, and/or oxidation of metals,including aluminum, stainless steel, and non-ferrous metals (e.g.,silver and copper). Suitable metal care agents include those describedin EP 2 100 949, WO 9426860 and WO 94/26859). In some embodiments, themetal care agent is a zinc salt. In some further embodiments, thecleaning compositions of the present invention comprise from about 0.1%to about 5% by weight of one or more metal care agent.

As indicated above, the cleaning compositions of the present inventionare formulated into any suitable form and prepared by any process chosenby the formulator, non-limiting examples of which are described in U.S.Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. In some embodiments in which a low pH cleaning composition isdesired, the pH of such composition is adjusted via the addition of anacidic material such as HCl.

The cleaning compositions disclosed herein of find use in cleaning asitus (e.g., a surface, dishware, or fabric). Typically, at least aportion of the situs is contacted with an embodiment of the presentcleaning composition, in neat form or diluted in a wash liquor, and thenthe situs is optionally washed and/or rinsed. For purposes of thepresent invention, “washing” includes but is not limited to, scrubbing,and mechanical agitation. In some embodiments, the cleaning compositionsare typically employed at concentrations of from about 500 ppm to about15,000 ppm in solution. When the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C. and, whenthe situs comprises a fabric, the water to fabric mass ratio istypically from about 1:1 to about 30:1.

EXPERIMENTAL

The following Examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); H₂O (water); HCl (hydrochloric acid); aa and AA (amino acid);by (base pair); kb (kilobase pair); kD (kilodaltons); nm (grams); μg andug (micrograms); mg (milligrams); ng (nanograms); μl and ul(microliters); ml (milliliters); mm (millimeters); nm (nanometers); μmand um (micrometer); M (molar); mM (millimolar); μM and uM (micromolar);U (units); V (volts); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₄₀₅ (opticaldensity at 405 nm); OD₆₀₀ (optical density at 600 nm); PAGE(polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphatebuffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]);LAS (lauryl sodium sulfonate); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); TAED(N,N,N′N′-tetraacetylethylenediamine); BES (polyesstersulfone); MES(2-morpholinoethanesulfonic acid, monohydrate; f.w. 195.24; Sigma #M-3671); CaCl₂ (calcium chloride, anhydrous; f.w. 110.99; Sigma #C-4901); SRI (Stain Removal Index), BMI (blood milk ink) and BMI PI(blood milk ink performance index), TCA (tricholoroacetic acid) and TCAPI (tricholoroacetic acid performance index).

In addition materials were obtained from some of the followinginstitutions: TIGR (The Institute for Genomic Research, Rockville, Md.);AATCC (American Association of Textile and Coloring Chemists); Amersham(Amersham Life Science, Inc. Arlington Heights, Ill.); Corning (CorningInternational, Corning, N.Y.); ICN (ICN Pharmaceuticals, Inc., CostaMesa, Calif.); Pierce (Pierce Biotechnology, Rockford, Ill.); Equest(Equest, Warwick International Group, Inc., Flintshire, UK); EMPA(Eidgenossische Material Prufungs and Versuch Anstalt, St. Gallen,Switzerland); CFT (Center for Test Materials, Vlaardingen, TheNetherlands); Amicon (Amicon, Inc., Beverly, Mass.); ATCC (American TypeCulture Collection, Manassas, Va.); Becton Dickinson (Becton DickinsonLabware, Lincoln Park, N.J.); Perkin-Elmer (Perkin-Elmer, Wellesley,Mass.); Rainin (Rainin Instrument, LLC, Woburn, Mass.); Eppendorf(Eppendorf AG, Hamburg, Germany); Waters (Waters, Inc., Milford, Mass.);Geneart (Geneart GmbH, Regensburg, Germany); Perseptive Biosystems(Perseptive Biosystems, Ramsey, Minn.); Molecular Probes (MolecularProbes, Eugene, Oreg.); BioRad (BioRad, Richmond, Calif.); Clontech(CLONTECH Laboratories, Palo Alto, Calif.); Cargill (Cargill, Inc.,Minneapolis, Minn.); Difco (Difco Laboratories, Detroit, Mich.); GIBCOBRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); NewBrunswick (New Brunswick Scientific Company, Inc., Edison, N.J.);Thermoelectron (Thermoelectron Corp., Waltham, Mass.); BMG (BMG Labtech,GmbH, Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster,Austria); Novagen (Novagen, Inc., Madison, Wis.); Novex (Novex, SanDiego, Calif.); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc.,Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad, Calif.);Sigma (Sigma Chemical Co., St. Louis, Mo.); DuPont Instruments(Asheville, N.Y.); Global Medical Instrumentation or GMI (Global MedicalInstrumentation; Ramsey, Minn.); MJ Research (MJ Research, Waltham,Mass.); Infors (Infors AG, Bottmingen, Switzerland); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann LaRoche, Inc., Nutley, N.J.); Agilent (Agilent Technologies, Palo Alto,Calif.); Merck (Merck & Co., Rahway, N.J.); Ion Beam Analysis Laboratory(Ion Bean Analysis Laboratory, The University of Surrey Ion Beam Centre(Guildford, UK); TOM (Terg-o-Meter); BMI (blood, milk, ink); BaChem(BaChem AG, Bubendorf, Switzerland); Molecular Devices (MolecularDevices, Inc., Sunnyvale, Calif.); Corning (Corning International,Corning, N.Y.); MicroCal (Microcal, Inc., Northhampton, Mass.); ChemicalComputing (Chemical Computing Corp., Montreal, Canada); NCBI (NationalCenter for Biotechnology Information); Beckman (Beckman-Coulter,Fullerton, Calif.); SeitzSchenk (SeitzSchenk Filtersystems GmbH, BadKreuznach, Germany); Pall (Pall Corp., East Hills, N.Y.); MalvernInstruments (Malvern Instruments, Inc., Worcestershire, UK), DNA 2.0(Menlo Park, Calif.), Molecular Devices (Sunnyvale, Calif.), Costar(Cambridge, Mass.).

EXAMPLE 1 Assays

In the following examples, various assays were used as set forth belowfor ease in reading. Any deviations from the protocols provided beloware indicated.

A. TCA Assay for Protein Content Determination in 96-Well MicrotiterPlates

For FNA (e.g., parent protease) and variants thereof, this assay wasstarted using filtered culture supernatant from microtiter plates grown3-4 days at 33° C. with shaking at 230 rpm and humidified aeration. Afresh 96-well flat bottom microtiter plate (MTP) was used for the assay.First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 50 μL offiltered culture broth was added. The light scattering/absorbance at 405nm (use 5 sec mixing mode in the plate reader) was then determined, inorder to provide the “blank” reading. For the test, 100 μL/well of 15%(w/v) trichloroacetic acid (TCA) was placed in the plates and incubatedbetween 5 and 30 min at room temperature. The lightscattering/absorbance at 405 nm (use 5 sec mixing mode in the platereader) was then determined.

For GG36 (e.g., parent protease) and variants thereof, this assay wasperformed using filtered culture supernatant from microtiter platesgrown approximately 3 days at 37° C. with shaking at 300 rpm andhumidified aeration. In this assay 100 μL of a 0.25 M HCl solution wasadded to each well of a 96-well flat bottom microtiter plate.Subsequently, 25 μL aliquots of the filtered culture supernatants(containing the proteases) were added to wells. The lightscattering/absorbance at 405 nm (using the 5 sec mixing mode in theplate reader) was then determined, in order to provide the “blank”reading. After this measurement, 100 μL of a 30% (w/v) TCA solution wasadded to each well and the microtiter plates were incubated between 5and 15 minutes at room temperature. Finally, the resulting lightscattering/absorbance at 405 nm (using the 5 sec mixing mode in theplate reader) was determined.

The equipment used was a Biomek FX Robot (Beckman Coulter) and aSpectraMAX (type 340; Molecular Devices) MTP Reader; the MTP's were fromCostar (type 9017). The equipment used was a Biomek FX Robot (BeckmanCoulter) and a SpectraMAX type 340 (Molecular Devices) MTP Reader; andthe MTPs were type 9017 (Costar).

The calculations were performed by subtracting the blank (no TCA) fromthe test reading with TCA to provide a relative measure of the proteincontent in the samples. If desired, a standard curve can be created bycalibrating the TCA readings with AAPF assays of clones with knownconversion factors. However, the TCA results are linear with respect toprotein concentration from 50 to 500 micrograms of protein per ml (ppm)and can thus be plotted directly against enzyme performance for thepurpose of choosing good-performing variants. The turbidity/lightscatter increase in the samples correlates to the total amount ofprecipitable protein in the culture supernatant.

B. Cleaning Performance Assays

The stain removal performance of reference serine proteases and variantsthereof on microswatches was determined on a microtiter plate (MTP)scale in commercially available TIDE® 2× Cold detergent. Heatinactivated TIDE® 2× Cold (off-the-shelf detergent) in which lack ofprotease activity was confirmed was used in the assays. Heatinactivation of commercial detergent formulas serves to destroy theenzymatic activity of any protein components while retaining theproperties of non-enzymatic components. Thus this method was suitablefor preparing commercially purchased detergents for use in testing theenzyme variants of the present invention. The reagents used were: 5 mMHEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium waterhardness: (CaCl₂:MgCl2.6H2O); 15000 grains per gallon (gpg) stockdiluted to 6 gpg. Two EMPA-116 BMI (blood/milk/ink) cotton swatchesprocessed by CFT were used per well. The microswatches were pre-washedin deionised water for 20 minutes at ambient temperature. After thepre-washing step, the swatches were put on top of paper towels to dry.The air-dried swatches were then punched using a ¼″ circular die on anexpulsion press. Finally two microswatches were put into each well of a96-well MTP vertically to expose the whole surface area (i.e. not flaton the bottom of the well). The working detergent solution is shown inTable 1-1.

TABLE 1-1 Working Detergent Solution Detergent Detergent Temp (C.) g/LpH Buffer gpg Protease TIDE ® 2X Cold 16 0.98 8 5 mM 6 FNA, HEPES GG36

The incubator was set 16° C. 10 μL samples from the master dilutionplate of ˜10 ppm enzyme was added to BMI 2-swatch plates with 190 μLworking detergent solutions listed above. The volume was adjusted togive final concentration of 0.5 ppm for variants in the assay plates.The plates were immediately transferred to iEMS incubator/shaker(Thermo/Labsystems); and incubated for 30 minutes with 1400 rpm shakingat given temperature. Following incubation, 100 μL of supernatant wastransferred into a new 96-well plate (Costar type 9017 used for readingreaction plates after incubation) and the absorbance was measured inSpectraMAX MTP Reader (type 340; Molecular Devices) at 405 nm and/or 600nm. Control wells, containing 2 microswatches and detergent without theaddition of protease samples were also included in the test. Themeasurement at 405 nm provides a higher value and tracks pigmentremoval, while the measurement at 600 nm tracks turbidity and cleaning.In this assay, the proteases hydrolyze the substrate and liberatepigment and insoluble particles from the substrate. Thus the rate ofturbidity is a measure of enzyme activity.

Calculation of the Stain Removal Activity

The absorbance value obtained was corrected for the blank value(substrate without enzyme), providing a measure of hydrolytic activity.For each sample (variant) the performance index was calculated. Theperformance index compares the performance of the variant (actual value)and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of the Langmuir equation of the standard enzyme.

Performance Index

The performance index compares the performance of the variant (actualvalue) and the parent protease (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of the binding curve (i.e., Langmuir equation) ofthe standard protease. A performance index (PI) that has a value >0.5for at least one property identifies a variant comprising at least onemutation that can be combined with one or more mutations to generateproteins having appropriate performance indices for one or moreproperties of interest other than or in addition to the property ofinterest for which a PI value of >0.5 was measured.

EXAMPLE 2 Bacillus subtilis GG36 Subtilisin Variants

In this example, experiments conducted to produce GG36 (also referred toherein as Bacillus lentus subtilisin) in B. subtilis are described.Transformation was performed as known in the art (See e.g., WO02/14490).

GG36 Protease Production in Bacillus subtilis

The expression plasmid pAC-GG36ci was assembled using the GG36codon-improved gene fused at the 8^(th) codon of the aprE signalsequence under the control of the consensus aprE promoter and the BPN′transcriptional terminator. In the sequence provided below (SEQ IDNO:2), bold and italicized font indicates consensus aprE promoter,standard font indicates the signal sequence, underlined font indicatesthe pro sequence, and bold font indicates DNA that encodes GG36 matureprotease, and underlined italicized font indicates BPN′ terminator. TheDNA sequence encoding the GG36 mature region (SEQ ID NO:4) is flanked byKpnI and XhoI restriction sites for cloning (see FIG. 4).

(SEQ ID NO: 2)

 

  

gtgagaagcaa aaaattgtggatcgtcgcgtcgaccgcattgctgatttctgttgcttttagctcatccatcgcatccgctgctgaagcaaaagaaaaatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaacaagttgaggcaaatgacgaggtagccattctctctgaggaagaggaggtcgaaattgaattgcttcatgaatttgaaacgattcctgttctgtccgttgagttaagcccagaagatgtggacgcgttagagctcgatccagctatttcttatattgaagagga tgcagaagtaactacaatggcgcaatcggtaccatggggaattagcagagtacaagccccagctgcacataaccgtggattgacaggttctggtgtaaaagttgctgtccttgataccggtatttccactcatccagacttaaatattcgtggtggagctagctttgtaccaggggaaccatccactcaagatggcaatggacatggcactcatgttgccggcacaatcgcggctcttaacaattcaattggtgttcttggcgtagcgccaagcgcagaactatacgctgttaaagtattaggagcaagcggttcaggctctgtcagctctattgcccaaggattggaatgggcagggaacaatggcatgcacgttgctaatcttagtttaggatctccttcgccaagtgccacacttgagcaagctgttaatagcgcgacttctagaggcgttcttgttgtagcggcctctggaaattcaggtgcaggctcaatcagctatccggcccgttatgcgaacgctatggcagtcggagctactgaccaaaacaacaaccgcgccagcttttcacagtatggcgcagggcttgacattgtcgcaccaggtgtaaacgtgcagagcacttacccaggttcaacatatgccagcttaaacggtacatcaatggctactcctcatgttgcaggtgcggctgcacttgttaaacaaaagaacccatcttggtccaatgtacaaatccgcaatcatcttaagaatacggcaactagcttaggaagcacaaacttgtatggaagcggacttgtcaatgcagaagctgcaactcgttaa aagcttaactcgagataaaaaaccggccttggccccgccggttttttat

The plasmid pAC-GG36ci shown in FIG. 4, was used for the expression ofGG36 protease in B. subtilis. The plasmid elements are as follows:pUB110=DNA fragment from plasmid pUB110 [McKenzie T., Hoshino T., TanakaT., Sueoka N. (1986) The Nucleotide Sequence of pUB110: Some SalientFeatures in Relation to Replication and Its Regulation. Plasmid15:93-103], pBR322=DNA fragment from plasmid pBR322 [Bolivar F,Rodriguez R L, Greene P J, Betlach M C, Heyneker H L, Boyer H W. (1977)Construction and characterization of new cloning vehicles. II. Amultipurpose cloning system. Gene 2:95-113], pC194=DNA fragment fromplasmid pC194 [Horinouchi S., Weisblum B. (1982) Nucleotide sequence andfunctional map of pC194, a plasmid that specifies induciblechloramphenicol resistance. J. Bacteriol 150:815-825].

Plasmid features are as follows: On for B. subtilis=origin ofreplication from pUB110, CAT=chloramphenicol resistance gene from pC194,pMB1 origin=origin of replication from pBR322, bla=beta-lactamase frompBR322, Short aprE promoter=consensus transcriptional promoter, SignalPeptide=signal peptide, Pro Peptide=GG36 pro region, GG36ci MaturePeptide=mature GG36 (replaced by the coding regions for each variantexpressed in this study), BPN′ Terminator=transcriptional terminatorfrom subtilisin BPN′.

The amino acid sequence of GG36 precursor protein is provided below (SEQID NO:3). In this sequence, bold indicates the mature GG36 protease(wildtype), which is also provided as SEQ ID NO:4:

(SEQ ID NO: 3) MRSKKLWIVASTALLISVAFSSSIASAAEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR* (SEQ ID NO: 4)AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATS LGSTNLYGSGLVNAEAATR*Design and Generation of Bacillus lentus Subtilisin (=GG36)Combinatorial Charge/Hydrophobic Library (CCHL)

The Bacillus lentus subtilisin combinatorial charge/hydrophobicitylibrary (CCHL) was designed by identifying seven well-distributed,surface-exposed amino-acids. These residues are S24, R45, S101, Q109,G118, T213, and L217. The number of the position of the substitution isby correspondence to the enumerated position in the BPN′ subtilisin(BPN′ numbering; FIG. 1). A 33-member combinatorial hydrophobic library(GH2-GH33) was created by making combinations of four possibilities ateach site: wild-type, glutamine (Q), glutamic acid (E), leucine (L) andarginine (R) as shown in Table 2-1.

The pAC-GG36ci plasmid containing the codon-improved GG36 gene was sentto DNA 2.0 Inc. (Menlo Park, Calif.) for the generation of the CHL. TheBacillus subtilis strain (genotype: ΔaprE, ΔnprE, ΔspoIIE,amyE::xylRPxylAcomK-phleo) was provided as the host strain for thetransformation of the DNA encoding the GG36 variant subtilisins.Variants were supplied as glycerol stocks in 96-well plates.

Table 2-2 shows the substitutions made in GG36 to create the variants.Table 2-1 also provides the difference in net charge and hydrophobicitybetween the variant GG36 and the wild-type.

Expression of Protease Variants

Bacillus subtilis clones containing GG36 or FNA expression vectors werereplicated with a steel 96-well replicator from glycerol stocks into96-well culture plates (BD, 353075) containing 200 μl of LB media+25μg/ml chloramphenicol, grown overnight at 37° C., 220 rpm in ahumidified enclosure. 200 μl from the overnight culture was used toinoculate 2000 μl defined media+25 μg/ml chloramphenicol in 5 ml plasticshake tubes. The cultivation media was an enriched semi-defined mediabased on MOPs buffer, with urea as major nitrogen source, glucose as themain carbon source, and supplemented with 1% soytone for robust cellgrowth. Shake tubes were incubated at 37° C., 220 rpm, for 60 hours.Following 60 hours, supernatants spun down in a centrifuge at greaterthan 8000×RCF. Solution was decanted into 15 ml polypropylene conicaltubes for storage. No further purification or concentration wasperformed. Supernatant stocks were formulated to 40% propylene glycolfor long-term stability and stored at 4° C.

TABLE 2-1 GG36 Combinatorial Charge/Hydrophobicity Library. Mutationsare Listed According to BPN′ Numbering. Charge/Hydrophobicity Changeswere Calculated Relative to GG36. Net Charge Kyte-Doolitle ChangeHydrophobicity Variant Relative Change relative # S24 R45 S101 Q109 G118T213 L217 Variant to GG36 to GG36 GG36 S24S R45R S101S Q109Q G118G T213TL217L S24S-R45R-S101S-Q109Q- 0 0 G118G-T213T-L217L GH-2 S24Q R45Q S101QQ109Q G118Q T213Q L217L S24Q-R45Q-S101Q-Q109Q- −1 −10.3G118Q-T213Q-L217L GH-3 S24S R45Q S101Q Q109Q G118Q T213Q L217LS24S-R45Q-S101Q-Q109Q- −1 −7.6 G118Q-T213Q-L217L GH-4 S24S R45R S101QQ109Q G118Q T213Q L217L S24S-R45R-S101Q-Q109Q- 0 −8.6 G118Q-T213Q-L217LGH-5 S24S R45R S101S Q109Q G118Q T213Q L217L S24S-R45R-S101S-Q109Q- 0−5.9 G118Q-T213Q-L217L GH-6 S24S R45R S101S Q109Q G118Q T213Q L217LS24S-R45R-S101S-Q109Q- 0 −5.9 G118Q-T213Q-L217L GH-7 S24S R45R S101SQ109Q G118G T213Q L217L S24S-R45R-S101S-Q109Q- 0 −2.8 G118G-T213Q-L217LGH-8 S24E R45Q S101Q Q109Q G118Q T213Q L217L S24E-R45Q-S101Q-Q109Q- −2−10.3 G118Q-T213Q-L217L GH-9 S24E R45E S101Q Q109Q G118Q T213Q L217LS24E-R45E-S101Q-Q109Q- −3 −10.3 G118Q-T213Q-L217L GH-10 S24E R45E S101EQ109Q G118Q T213Q L217L S24E-R45E-S101E-Q109Q- −4 −10.3G118Q-T213Q-L217L GH-11 S24E R45E S101E Q109E G118Q T213Q L217LS24E-R45E-S101E-Q109E- −5 −10.3 G118Q-T213Q-L217L GH-12 S24E R45E S101EQ109E G118E T213Q L217L S24E-R45E-S101E-Q109E- −6 −10.3G118E-T213Q-L217L GH-13 S24E R45E S101E Q109E G118E T213E L217LS24E-R45E-S101E-Q109E- −7 −10.3 G118E-T213E-L217L GH-14 S24L R45Q S101QQ109Q G118Q T213Q L217L S24L-R45Q-S101Q-Q109Q- −1 −3 G118Q-T213Q-L217LGH-15 S24L R45L S101Q Q109Q G118Q T213Q L217L S24L-R45L-S101Q-Q109Q- −14.3 G118Q-T213Q-L217L GH-16 S24L R45L S101L Q109Q G118Q T213Q L217LS24L-R45L-S101L-Q109Q- −1 11.6 G118Q-T213Q-L217L GH-17 S24L R45L S101LQ109L G118Q T213Q L217L S24L-R45L-S101L-Q109L- −1 18.9 G118Q-T213Q-L217LGH-18 S24L R45L S101L Q109L G118L T213Q L217L S24L-R45L-S101L-Q109L- −126.2 G118L-T213Q-L217L GH-19 S24L R45L S101L Q109L G118L T213L L217LS24L-R45L-S101L-Q109L- −1 33.5 G118L-T213L-L217L GH-20 S24R R45Q S101QQ109Q G118Q T213Q L217L S24R-R45Q-S101Q-Q109Q- 0 −11.3 G118Q-T213Q-L217LGH-21 S24R R45R S101Q Q109Q G118Q T213Q L217L S24R-R45R-S101Q-Q109Q- 1−12.3 G118Q-T213Q-L217L GH-22 S24R R45R S101R Q109Q G118Q T213Q L217LS24R-R45R-S101R-Q109Q- 2 −13.3 G118Q-T213Q-L217L GH-23 S24R R45R S101RQ109R G118Q T213Q L217L S24R-R45R-S101R-Q109R- 3 −14.3 G118Q-T213Q-L217LGH-24 S24R R45R S101R Q109R G118R T213Q L217L S24R-R45R-S101R-Q109R- 4−15.3 G118R-T213Q-L217L GH-25 S24R R45R S101R Q109R G118R T213R L217LS24R-R45R-S101R-Q109R- 5 −16.3 G118R-T213R-L217L GH-26 S24E R45R S101RQ109R G118R T213R L217L S24E-R45R-S101R-Q109R- 3 −15.3 G118R-T213R-L217LGH-27 S24E R45E S101R Q109R G118R T213R L217L S24E-R45E-S101R-Q109R- 1−14.3 G118R-T213R-L217L GH-28 S24E R45E S101E Q109R G118R T213R L217LS24E-R45E-S101E-Q109R- −1 −13.3 G118R-T213R-L217L GH-29 S24E R45E S101EQ109E G118R T213R L217L S24E-R45E-S101E-Q109E- −3 −12.3G118R-T213R-L217L GH-30 S24E R45E S101E Q109E G118E T213R L217LS24E-R45E-S101E-Q109E- −5 −11.3 G118E-T213R-L217L GH-31 S24E R45E S101EQ109E G118E T213E L217L S24E-R45E-S101E-Q109E- −7 −10.3G118E-T213E-L217L GH-32 S24Q R45Q S101Q Q109Q G118Q T213Q L217QS24Q-R45Q-S101Q-Q109Q- −1 −17.6 G118Q-T213Q-L217Q GH-33 S24Q R45Q S101QQ109Q G118Q T213Q L217E S24Q-R45Q-S101Q-Q109Q- −2 −17.6G118Q-T213Q-L217E

EXAMPLE 3 Bacillus subtilis FNA Subtilisin Variants

FNA Protease Production in B. subtilis

In this example, experiments conducted to produce FNA (also referred toherein as Bacillus subtilis subtilisin BPN′-Y217L (=FNA)) in B. subtilisare described. Transformation was performed as known in the art (Seee.g., WO 02/14490).

The expression plasmid pAC-FNAre was assembled using the FNA gene, fusedat the 8^(th) codon of the aprE signal sequence, under the control ofthe consensus aprE promoter and BPN′ transcriptional terminator. In thesequence provided below (SEQ ID NO:5), bold and italicized fontindicates consensus aprE promoter, standard font indicates the signalsequence, underlined font indicates the pro sequence, bold fontindicates DNA that encodes FNA mature protease, and underlineditalicized font indicates BPN′ terminator. The FNA mature regioncontains the KpnI and XhoI restriction sites for cloning (see FIG. 5).

(SEQ ID NO: 5)

        

  

    

gtgag aagcaaaaaattgtggatcagtttgctgtttgctttagcgttaatctttacgatggcgttcggcagcacatccagcgcgcaggctgcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaagaagaaagacgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcagctagcgctacattaaacgaaaaagctgtaaaagaattgaaaaaagacccgagcgtcgcttacgttgaagaagatca cgtagcacacgcgtacgcgcagtccgtgccatatggcgtatcacaaattaaagcccctgctctgcactctcaaggctacaccggttcaaatgttaaagtagcggttatcgacagcggtatcgattcttctcatccagatcttaaagtagcaggcggagccagcatggttccttctgaaacaaatcctttccaagacaacaactctcacggaacacacgttgctggtaccgttgcggctcttaataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagttctcggcgccgacggttccggccaatacagctggatcattaacggaatcgagtgggcgatcgcaaacaatatggacgttattaacatgagcctcggcggaccgtccggttctgctgctttaaaagcggcagttgataaagccgttgcatccggcgtcgtagtcgttgcggcagccggcaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcattgcagtaggcgctgtcgacagcagcaaccaaagagcatctttctcaagcgtaggacctgagctcgatgtcatggcacctggcgtatctatccaaagcacgcttcctggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagccgcggctttgattctttctaagcacccgaactggacaaacactcaagtccgcagctctctagaaaacaccactacaaaacttggtgattctttctactatggaaaagggctgatcaatgtacaggcggcagctcagtaa aactcgagataaaaaaccggccttggccccgccggttttttat .

The plasmid pAC-FNAre as shown in FIG. 5, was used for the expression ofFNA protease in B. subtilis. The plasmid elements are as follows:pUB110=DNA fragment from plasmid pUB110 [McKenzie T., Hoshino T., TanakaT., Sueoka N. (1986) The Nucleotide Sequence of pUB110: Some SalientFeatures in Relation to Replication and Its Regulation. Plasmid15:93-103], pBR322=DNA fragment from plasmid pBR322 [Bolivar F,Rodriguez R L, Greene P J, Betlach M C, Heyneker H L, Boyer H W. (1977)Construction and characterization of new cloning vehicles. II. Amultipurpose cloning system. Gene 2:95-113], pC194=DNA fragment fromplasmid pC194 [Horinouchi S., Weisblum B. (1982) Nucleotide sequence andfunctional map of pC194, a plasmid that specifies induciblechloramphenicol resistance. J. Bacteriol 150:815-825].

Plasmid features are as follows: On for B. subtilis=origin ofreplication from pUB110, CAT=chloramphenicol resistance gene from pC194,pMB1 origin=origin of replication from pBR322, bla=beta-lactamase frompBR322, Short aprE promoter=consensus transcriptional promoter, SignalPeptide=signal peptide [specify], Pro Peptide=FNA pro region, FNA MaturePeptide=mature FNA (replaced by the coding regions for each variantexpressed in this study), BPN′ Terminator=transcriptional terminatorfrom subtilisin BPN′.

The amino acid sequence of FNA precursor protein is provided below (SEQID NO:6). In this sequence, bold indicates the mature FNA protease(wildtype), which is also provided as SEQ ID NO:7.

(SEQ ID NO: 6) MRSKKLWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVISEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGALNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ* (SEQ ID NO: 7)AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGALNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ*Design and Generation of subtilisin BPN′-Y217L (=FNA) CombinatorialCharge/Hydrophobic Libraries (CHL)

The subtilisin BPN′-Y217L combinatorial charge hydrophobic library wasdesigned by identifying seven well-distributed, surface-exposed aminoacids. These residues are S24, A45, S101, N109, N118, K213, and L217. A32-member combinatorial hydrophobic library (FH2-FH33) was created bymaking combinations of four possibilities at each site: wild-type,glutamine (Q), glutamic acid (E), leucine (L) and arginine (R) as shownin Table 3-1.

The pAC-FNAre plasmid containing the FNA gene was sent to DNA 2.0 Inc.(Menlo Park, Calif.) for the generation of the Combinatorial HydrophobicLibraries. The Bacillus subtilis strain (genotype: ΔaprE, ΔnprE,ΔspoIIE, amyE::xylRPxylAcomK-phleo) was provided for the transformationsof the DNA encoding the FNA variant substitutions. Variants weresupplied as glycerol stocks in 96-well plates.

Table 3-1 shows the substitutions made in FNA to create the variants.Table 3-1 also provides the difference in net charge and hydrophobicitybetween the variant FNA and the wild-type.

Expression of Protease Variants

Bacillus subtilis clones containing GG36 or FNA expression vectors werereplicated with a steel 96-well replicator from glycerol stocks into96-well culture plates (Becton Dickinson, 353075) containing 200 μl ofLB media+25 μg/ml chloramphenicol, grown overnight at 37° C., 220 rpm ina humidified enclosure. 200 μl from the overnight culture was used toinoculate 2000 μl defined media+25 μg/ml chloramphenicol in 5 ml plasticshake tubes. The cultivation media was an enriched semi-defined mediabased on MOPs buffer, with urea as major nitrogen source, glucose as themain carbon source, and supplemented with 1% soytone for robust cellgrowth. Shake tubes were incubated at 37° C., 220 rpm, for 60 hours.Following 60 hours, supernatants spun down in a centrifuge at greaterthan 8000×RCF. Solution was decanted into 15 ml polypropylene conicaltubes for storage. No further purification or concentration wasperformed. Supernatant stocks were formulated to 40% propylene glycolfor long-term stability and stored at

TABLE 3-1 FNA Combinatorial Charge/Hydrophobicity Library. Mutations arelisted according to BPN′ numbering. Charge/Hydrophobicity changes werecalculated relative to FNA. Net Charge Kyte-Doolitle ChangeHydrophobicity Variant Relative Change relative # S24 A45 S101 N109 N118K213 L217 Variant to FNA to FNA FNA S24S A45A S101S N109N N118N K213KL217L S24S-A45A-S101S-N109N-N118N- 0 0 K213K-L217L FH-2 S24Q A45Q S101QN109Q N118Q K213Q L217L S24Q-A45Q-S101Q-N109Q-N118Q- −1 −10.3K213Q-L217L FH-3 S24S A45Q S101Q N109Q N118Q K213Q L217LS24S-A45Q-S101Q-N109Q-N118Q- −1 −7.6 K213Q-L217L FH-4 S24S A45A S101QN109Q N118Q K213Q L217L S24S-A45A-S101Q-N109Q-N118Q- −1 −2.3 K213Q-L217LFH-5 S24S A45A S101S N109Q N118Q K213Q L217LS24S-A45A-S101S-N109Q-N118Q- −1 0.4 K213Q-L217L FH-6 S24S A45A S101SN109N N118Q K213Q L217L S24S-A45A-S101S-N109N-N118Q- −1 0.4 K213Q-L217LFH-7 S24S A45A S101S N109N N118N K213Q L217LS24S-A45A-S101S-N109N-N118N- −1 0.4 K213Q-L217L FH-8 S24E A45Q S101QN109Q N118Q K213Q L217L S24E-A45Q-S101Q-N109Q-N118Q- −2 −10.3K213Q-L217L FH-9 S24E A45E S101Q N109Q N118Q K213Q L217LS24E-A45E-S101Q-N109Q-N118Q- −3 −10.3 K213Q-L217L FH-10 S24E A45E S101EN109Q N118Q K213Q L217L S24E-A45E-S101E-N109Q-N118Q- −4 −10.3K213Q-L217L FH-11 S24E A45E S101E N109E N118Q K213Q L217LS24E-A45E-S101E-N109E-N118Q- −5 −10.3 K213Q-L217L FH-12 S24E A45E S101EN109E N118E K213Q L217L S24E-A45E-S101E-N109E-N118E- −6 −10.3K213Q-L217L FH-13 S24E A45E S101E N109E N118E K213E L217LS24E-A45E-S101E-N109E-N118E- −7 −10.3 K213E-L217L FH-14 S24L A45Q S101QN109Q N118Q K213Q L217L S24L-A45Q-S101Q-N109Q-N118Q- −1 −3 K213Q-L217LFH-15 S24L A45L S101Q N109Q N118Q K213Q L217LS24L-A45L-S101Q-N109Q-N118Q- −1 4.3 K213Q-L217L FH-16 S24L A45L S101LN109Q N118Q K213Q L217L S24L-A45L-S101L-N109Q-N118Q- −1 11.6 K213Q-L217LFH-17 S24L A45L S101L N109L N118Q K213Q L217LS24L-A45L-S101L-N109L-N118Q- −1 18.9 K213Q-L217L FH-18 S24L A45L S101LN109L N118L K213Q L217L S24L-A45L-S101L-N109L-N118L- −1 26.2 K213Q-L217LFH-19 S24L A45L S101L N109L N118L K213L L217LS24L-A45L-S101L-N109L-N118L- −1 33.5 K213L-L217L FH-20 S24R A45Q S101QN109Q N118Q K213Q L217L S24R-A45Q-S101Q-N109Q-N118Q- 0 −11.3 K213Q-L217LFH-21 S24R A45R S101Q N109Q N118Q K213Q L217LS24R-A45R-S101Q-N109Q-N118Q- 1 −12.3 K213Q-L217L FH-22 S24R A45R S101RN109Q N118Q K213Q L217L S24R-A45R-S101R-N109Q-N118Q- 2 −13.3 K213Q-L217LFH-23 S24R A45R S101R N109R N118Q K213Q L217LS24R-A45R-S101R-N109R-N118Q- 3 −14.3 K213Q-L217L FH-24 S24R A45R S101RN109R N118R K213Q L217L S24R-A45R-S101R-N109R-N118R- 4 −15.3 K213Q-L217LFH-25 S24R A45R S101R N109R N118R K213R L217LS24R-A45R-S101R-N109R-N118R- 5 −16.3 K213R-L217L FH-26 S24E A45R S101RN109R N118R K213R L217L S24E-A45R-S101R-N109R-N118R- 3 −15.3 K213R-L217LFH-27 S24E A45E S101R N109R N118R K213R L217LS24E-A45E-S101R-N109R-N118R- 1 −14.3 K213R-L217L FH-28 S24E A45E S101EN109R N118R K213R L217L S24E-A45E-S101E-N109R-N118R- −1 −13.3K213R-L217L FH-29 S24E A45E S101E N109E N118R K213R L217LS24E-A45E-S101E-N109E-N118R- −3 −12.3 K213R-L217L FH-30 S24E A45E S101EN109E N118E K213R L217L S24E-A45E-S101E-N109E-N118E- −5 −11.3K213R-L217L FH-31 S24E A45E S101E N109E N118E K213E L217LS24E-A45E-S101E-N109E-N118E- −7 −10.3 K213E-L217L FH-32 S24Q A45Q S101QN109Q N118Q K213Q L217Q S24Q-A45Q-S101Q-N109Q-N118Q- −1 −17.6K213Q-L217Q FH-33 S24Q A45Q S101Q N109Q N118Q K213Q L217ES24Q-A45Q-S101Q-N109Q-N118Q- −2 −17.6 K213Q-L217E

EXAMPLE 4 Evaluation of Stain Removal and Relative Expression

This Example describes the testing of GG36 and FNA combinatorialhydrophobic library variants in a BMI microswatch assay. The methodsprovided in Example 1 were used. The results shown in Tables 4-1 (GG36),and 4-2 (FNA) are performance indices in which the performance of thevariant is compared to the respective parent for relative proteinexpression (TCA PI) and stain removal activity (BMI PI). Those variantswith a performance index greater than 0.5 (PI>0.5) have improvedperformance. Performance index less than or equal to 0.05 were set at0.05 and are indicated in bold italics. ND indicates not determined.

TABLE 4-1 Relative Expression and Stain Removal Performance of GG36Combinatorial/Charge Hydrophobicity Library Variants. Mutations areListed According to BPN′ Numbering. Performance Index andCharge/Hydrophobicity Changes Calculated Relative to GG36 (parent) NetCharge Kyte-Doolitle Change Hydrophobicity Relative Change relative TCABMI Variant # Variant to GG36 to GG36 PI PI GG36 S24S-R45R- 0 0 1.001.00 S101S-Q109Q- G118G-T213T- L217L GH-2 S24Q-R45Q- −1 −10.3 1.03 0.06S101Q-Q109Q- G118Q-T213Q- L217L GH-3 S24S-R45Q- −1 −7.6 1.05 0.42S101Q-Q109Q- G118Q-T213Q- L217L GH-4 S24S-R45R- 0 −8.6 0.87 0.23S101Q-Q109Q- G118Q-T213Q- L217L GH-5 S24S-R45R- 0 −5.9 1.00 0.05S101S-Q109Q- G118Q-T213Q- L217L GH-6 S24S-R45R- 0 −5.9 ND NDS101S-Q109Q- G118Q-T213Q- L217L GH-7 S24S-R45R- 0 −2.8 0.79 0.38S101S-Q109Q- G118G-T213Q- L217L GH-8 S24E-R45Q- −2 −10.3 1.01 0.47S101Q-Q109Q- G118Q-T213Q- L217L GH-9 S24E-R45E- −3 −10.3 0.89 0.26S101Q-Q109Q- G118Q-T213Q- L217L GH-10 S24E-R45E- −4 −10.3 1.28 0.04S101E-Q109Q- G118Q-T213Q- L217L GH-11 S24E-R45E- −5 −10.3 1.09 0.78S101E-Q109E- G118Q-T213Q- L217L GH-12 S24E-R45E- −6 −10.3 1.10 0.29S101E-Q109E- G118E-T213Q- L217L GH-13 S24E-R45E- −7 −10.3 1.06 0.71S101E-Q109E- G118E-T213E- L217L GH-14 S24L-R45Q- −1 −3 0.96 0.37S101Q-Q109Q- G118Q-T213Q- L217L GH-15 S24L-R45L- −1 4.3 0.99 0.52S101Q-Q109Q- G118Q-T213Q- L217L GH-16 S24L-R45L- −1 11.6 0.88 0.08S101L-Q109Q- G118Q-T213Q- L217L GH-17 S24L-R45L- −1 18.9 0.90 0.40S101L-Q109L- G118Q-T213Q- L217L GH-18 S24L-R45L- −1 26.2 0.96 0.09S101L-Q109L- G118L-T213Q- L217L GH-19 S24L-R45L- −1 33.5 1.06

S101L-Q109L- G118L-T213L- L217L GH-20 S24R-R45Q- 0 −11.3 1.05 0.56S101Q-Q109Q- G118Q-T213Q- L217L GH-21 S24R-R45R- 1 −12.3 0.78 0.21S101Q-Q109Q- G118Q-T213Q- L217L GH-22 S24R-R45R- 2 −13.3 0.74 0.19S101R-Q109Q- G118Q-T213Q- L217L GH-23 S24R-R45R- 3 −14.3 0.75 0.15S101R-Q109R- G118Q-T213Q- L217L GH-24 S24R-R45R- 4 −15.3 0.68 0.07S101R-Q109R- G118R-T213Q- L217L GH-25 S24R-R45R- 5 −16.3 1.09

S101R-Q109R- G118R-T213R- L217L GH-26 S24E-R45R- 3 −15.3 0.90

S101R-Q109R- G118R-T213R- L217L GH-27 S24E-R45E- 1 −14.3 0.79

S101R-Q109R- G118R-T213R- L217L GH-28 S24E-R45E- −1 −13.3 0.87 0.06S101E-Q109R- G118R-T213R- L217L GH-29 S24E-R45E- −3 −12.3 0.92 0.31S101E-Q109E- G118R-T213R- L217L GH-30 S24E-R45E- −5 −11.3 0.93 0.47S101E-Q109E- G118E-T213R- L217L GH-31 S24E-R45E- −7 −10.3 1.01 0.40S101E-Q109E- G118E-T213E- L217L GH-32 S24Q-R45Q- −1 −17.6 1.10 0.07S101Q-Q109Q- G118Q-T213Q- L217Q GH-33 S24Q-R45Q- −2 −17.6 1.11 0.47S101Q-Q109Q- G118Q-T213Q- L217E

TABLE 4-2 Relative Expression and Stain Removal Performance of FNACombinatorial Charge/Hydrophobicity Library Variants. Mutations areListed According to BPN′ Numbering. Performance Index andCharge/Hydrophobicity Changes Calculated Relative to FNA (parent). NetCharge Kyte-Doolitle Change Hydrophobicity Relative Change relative TCABMI Variant # Variant to FNA to FNA PI PI FNA S24S-A45A- 0 0 1.00 1.00S101S-N109N- N118N-K213K- L217L FH-2 S24Q-A45Q- −1 −10.3 1.62 0.93S101Q-N109Q- N118Q-K213Q- L217L FH-3 S24S-A45Q- −1 −7.6 2.08 1.00S101Q-N109Q- N118Q-K213Q- L217L FH-4 S24S-A45A- −1 −2.3 1.46 1.01S101Q-N109Q- N118Q-K213Q- L217L FH-5 S24S-A45A- −1 0.4 1.29 0.95S101S-N109Q- N118Q-K213Q- L217L FH-6 S24S-A45A- −1 0.4 1.70

S101S-N109N- N118Q-K213Q- L217L FH-7 S24S-A45A- −1 0.4 1.95 0.95S101S-N109N- N118N-K213Q- L217L FH-8 S24E-A45Q- −2 −10.3 1.55 0.90S101Q-N109Q- N118Q-K213Q- L217L FH-9 S24E-A45E- −3 −10.3 1.37 0.88S101Q-N109Q- N118Q-K213Q- L217L FH-10 S24E-A45E- −4 −10.3 1.52 0.68S101E-N109Q- N118Q-K213Q- L217L FH-11 S24E-A45E- −5 −10.3 2.72 0.81S101E-N109E- N118Q-K213Q- L217L FH-12 S24E-A45E- −6 −10.3 1.55 0.56S101E-N109E- N118E-K213Q- L217L FH-13 S24E-A45E- −7 −10.3 1.56 0.42S101E-N109E- N118E-K213E- L217L FH-14 S24L-A45Q- −1 −3 1.27 1.02S101Q-N109Q- N118Q-K213Q- L217L FH-15 S24L-A45L- −1 4.3 1.68 0.98S101Q-N109Q- N118Q-K213Q- L217L FH-16 S24L-A45L- −1 11.6 1.47 0.94S101L-N109Q- N118Q-K213Q- L217L FH-17 S24L-A45L- −1 18.9 1.17 0.89S101L-N109L- N118Q-K213Q- L217L FH-18 S24L-A45L- −1 26.2 1.11 0.23S101L-N109L- N118L-K213Q- L217L FH-19 S24L-A45L- −1 33.5 1.19 0.20S101L-N109L- N118L-K213L- L217L FH-20 S24R-A45Q- 0 −11.3 1.31 1.01S101Q-N109Q- N118Q-K213Q- L217L FH-21 S24R-A45R- 1 −12.3 1.29 0.75S101Q-N109Q- N118Q-K213Q- L217L FH-22 S24R-A45R- 2 −13.3 1.28 0.46S101R-N109Q- N118Q-K213Q- L217L FH-23 S24R-A45R- 3 −14.3 1.21 0.33S101R-N109R- N118Q-K213Q- L217L FH-24 S24R-A45R- 4 −15.3 1.11 0.28S101R-N109R- N118R-K213Q- L217L FH-25 S24R-A45R- 5 −16.3 0.98 0.23S101R-N109R- N118R-K213R- L217L FH-26 S24E-A45R- 3 −15.3 1.22 0.37S101R-N109R- N118R-K213R- L217L FH-27 S24E-A45E- 1 −14.3 1.23 0.65S101R-N109R- N118R-K213R- L217L FH-28 S24E-A45E- −1 −13.3 1.55 0.89S101E-N109R- N118R-K213R- L217L FH-29 S24E-A45E- −3 −12.3 1.56 0.84S101E-N109E- N118R-K213R- L217L FH-30 S24E-A45E- −5 −11.3 1.48 0.82S101E-N109E- N118E-K213R- L217L FH-31 S24E-A45E- −7 −10.3 2.89 0.65S101E-N109E- N118E-K213E- L217L FH-32 S24Q-A45Q- −1 −17.6 1.55 1.07S101Q-N109Q- N118Q-K213Q- L217Q FH-33 S24Q-A45Q- −2 −17.6 1.68 1.07S101Q-N109Q- N118Q-K213Q- L217E

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. Those of skill in the art readily appreciate thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein. The compositions and methods described herein arerepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. It is readilyapparent to one skilled in the art that varying substitutions andmodifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by herein.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notexcised material is specifically recited herein.

We claim:
 1. A method of cleaning, said method comprising the steps of:a) contacting a surface and/or an article comprising a fabric with asubtilisin protease variant of a parent Bacillus subtilisin, whereinsaid subtilisin variant is a mature form having proteolytic activity andcomprising a substitution at two or more positions selected frompositions 24, 45, 101, 109, 118, 213 and 217, wherein said positions arenumbered by correspondence with the amino acid sequence of B.amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1, and whereinsaid parent has the sequence of SEQ ID NO:4 or 7; and b) optionallywashing and/or rinsing said surface or article.
 2. The method of claim1, wherein the subtilisin protease variant has a relative proteinexpression level performance index (TCA PI) and/or a stain removalactivity performance index (BMI PI) that is greater or equal to 0.5. 3.The method of claim 1, wherein the subtilisin protease variant has arelative protein expression level performance index (TCA PI) and/or astain removal activity performance index (BMI PI) that is greater than1.0.
 4. The method of claim 1, wherein said Bacillus subtilisin is GG36,and wherein said substitution at two or more positions is selected from:S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L, S101R,Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L, T213R,T213E, L217Q, and L217E, wherein the positions correspond to thepositions of BPN' subtilisin of SEQ ID NO:1.
 5. The method of claim 1,wherein said Bacillus subtilisin is FNA, and wherein said substitutionat two or more positions is selected from: S24Q, S24E, S24L, S24R, A45Q,A45E, A45L, A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L,N109R, K213Q, K213E, K213L, K213R, L217Q, L217E, wherein the positionscorrespond to the positions of BPN' subtilisin of SEQ ID NO:
 1. 6. Themethod of claim 1, wherein said Bacillus subtilisin is GG36, and whereinsaid substitution at two or more positions is selected fromS24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q,G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-GI18Q-T213Q,S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101 E-Q109E-G118Q-T213Q,S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E,S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q,S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q,S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L,S24R-R45Q-S 101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q,S24R-S101R-GII8Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q, S24R-S101R-Q109R-G 118R-T213Q, S24R-S 101R-Q 109R-G 118R-T213R, S24E-S 101 R-Q109R-G 118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R,S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R,S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E,S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, and S24Q-R45Q-S101 Q-G118Q-T213Q-L217E, wherein the positions correspond to the positions ofBPN' subtilisin of SEQ ID NO:
 1. 7. The method of claim 1, wherein saidBacillus subtilisin is GG36, and said subtilisin variant is a matureform having proteolytic activity and comprising the substitution T213Q,wherein said position corresponds to the amino acid sequence of B.amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:
 1. 8. Themethod of claim 1, wherein said Bacillus subtilisin is FNA, and whereinsaid substitution at two or more positions is selected fromS24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-NI18Q-K213Q,S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q,S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q,S24E-A45E-S101 E-N109Q-N118Q-K213Q, S24E-A45E-S101 E-N109E-N118Q-K213Q,S24E-A45E-S101E-N109E-N118E-K213Q, S24E-A45E-S101E-N109E-N118E-K213E,S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q,S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q,S24L-A45L-S101L-N109L-N118L-K213Q, S24L-A45L-S101L-N109L-N118L-K213L,S24R-A45Q-S101Q-N109Q-NII8Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q,S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K2130,S24R-A45R-S101R-N109R-N118R-K213Q, S24R-A45R-S101R-N109R-N118R-K213R,S24E-A45R-S101R-N109R-NII8R-K213R, S24E-A45E-S101R-N109R-N118R-K213R,S24E-A45E-S101E-N109R-N118R-K213R, S24E-A45E-S101E-N109E-N118R-K213R,S24E-A45E-S101 E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E,S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, andS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positionscorrespond to the positions of BPN' subtilisin of SEQ ID NO:
 1. 9. Themethod of claim 1, wherein said variant has proteolytic activity andcomprises the substitution K213Q, wherein said position is numbered bycorrespondence with the amino acid sequence of B. amyloliquefacienssubtilisin BPN' set forth as SEQ ID NO:
 1. 10. The method of claim 1,further comprising contact the surface and/or an article comprising afabric with one or more additional enzymes or enzyme derivativesselected from hemicellulases, cellulases, peroxidases, proteases,metalloproteases, xylanases, lipases, phospholipases, esterases,perhydrolasess, cutinases, pectinases, pectate lyases, mannanases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,B-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof.